A fungal effector promotes infection via stabilizing a negative regulatory factor of chloroplast immunity

Xiao, Kunqin; Yang, Feng; Cui, Wenjing; Li, Anmo; Rollins, Jeffrey A.; Guo, Jinxin; Sun, Xinhua; Wang, Fengting; Wang, Xiaojie; Xu, Xun; Zhang, Yanhua; Zhang, Xianghui; Liu, Jinliang; Pan, Hongyu

doi:10.1038/s41467-025-62326-4

Download PDF

Article
Open access
Published: 29 July 2025

A fungal effector promotes infection via stabilizing a negative regulatory factor of chloroplast immunity

Nature Communications volume 16, Article number: 6970 (2025) Cite this article

3833 Accesses
2 Altmetric
Metrics details

Subjects

Abstract

Chloroplasts are crucial players in immunity and photosynthesis. However, how chloroplasts arrange the transition between photosynthesis and immunity and how pathogens manipulate this transition remains elusive. Here we report an effector SsCm1 from Sclerotinia sclerotiorum, one of devastating phytopathogenic fungi, inhibits chloroplast immunity and resistance to pathogens, and alleviates photoinhibition in immune state. This is accomplished through stabilizing the conserved chloroplast protein MORF2, which is degraded during immunization and is a suppressor of photoinhibition, cell death, and chloroplast immunity. Overexpression of SsCm1 or MORF2 in plants represses basic immunity and resistance to pathogens, whereas deletion of SsCm1 reduces S. sclerotiorum virulence. Notably, SsCm1 possesses no chorismate mutase activity, which is different from the previously reported Cm effectors. This work reveals a strategy to fine-tune growth-defense balance in chloroplasts by manipulating MORF2, and a pathogenic strategy to subvert the process and promote infection via enzymatically nonfunctional effector stabilizing MORF2.

An effector essential for virulence of necrotrophic fungi targets plant HIRs to inhibit host immunity

Article Open access 30 October 2024

Epidermal chloroplasts are defense-related motile organelles equipped with plant immune components

Article Open access 20 May 2021

Chloroplast elongation factors break the growth–immunity trade-off by simultaneously promoting yield and defence

Article 19 September 2024

Introduction

Plants perceive threats from pathogens through complex and ingenious mechanisms: the pattern-recognition receptors (PRRs) located on the plasma membrane detect pathogen-associated molecular patterns (PAMPs) and activate basic immunity (PAMP-triggered immunity, PTI); intracellular nucleotide-binding leucine-rich repeat receptors recognize pathogen effectors to activate more robust effector-triggered immunity (ETI)¹. Both PTI and ETI are involved in overlapping downstream immune responses, including activation of mitogen-activated protein kinases (MAPKs), calcium-mediated signaling, reactive oxygen species (ROS) burst, transcriptional reprograming, callose deposition, and biosynthesis of salicylic acid (SA)^2,3. Chloroplasts play vital roles in these immune responses and are crucial participants in plant-pathogen interactions^4,5,6,7.

Chloroplasts are emerging as hubs in integrating environmental stimuli and determinants of biotic or abiotic stress responses^7,8,9. The current views ascribe fundamental function to chloroplasts in orchestrating defense responses: upon plasma membrane sensing biological threats, signals are quickly relayed to chloroplasts through the plasma membrane/chloroplast dual-located proteins or calcium signal^5,7. Then, chloroplasts produce calcium signatures, ROS, the by-products of tetrapyrrole pathway and communicate with the nucleus via retrograde signaling (RS)^{7,10,11,12,13}. This signaling results in the expression of defense-related genes, such as genes involved in SA biosynthesis and encoding WRKY transcription factors¹⁴. When the threat persists or is fatal, MPK3/MPK6 actively inhibits photosynthesis to promote ROS accumulation in chloroplasts¹⁵. These processes represent the transformation from growth to defense, including immune transcriptional reprograming, SA biosynthesis, localized cell death, and resistance to pathogens^16,17,18,19. Note that these changes come at the expense of the photosynthetic function of chloroplasts, including global down-regulation of photosynthetic genes, photoinhibition, and damage of photosystem II (PSII), which are essential for chloroplast immunity^{20,21,22,23,24}. Therefore, chloroplasts require precise control to rapidly convert to the immune state upon pathogen infection while maintaining the photosynthetic state under normal conditions. How chloroplasts switch between these two opposite states and which components are responsible for this switch are unclear.

Consistent with the prominent role of chloroplasts in immunity and the arms race between pathogens and hosts, this organelle is a prime target for pathogen manipulation^4,5,6. Convergently evolved effectors of pathogens from different kingdoms target chloroplasts and impair SA-dependent immunity to promote pathogenicity⁵. Virulent Pseudomonas syringae secretes multiple effectors that cooperate to reprogram the expression of photosynthesis-associated nuclear genes (PhANGs) and target the chloroplast to prevent the chloroplastic ROS burst by impairing PSI⁶. The effector Pst_12806 reduces the levels of photosynthesis and chloroplast-derived ROS to facilitate pathogen survival by targeting the chloroplastic cytochrome b6-f complex²⁵. Similarly, Sclerotinia sclerotiorum also employs virulence factors, including oxalate and SsITL to increase chloroplast non-photochemical quenching (NPQ) and perturb immune function of the chloroplastic calcium-sensing receptor, respectively, to suppress host defense^26,27,28. However, it is unclear how pathogens can manipulate the fine-tuned process of chloroplast switching from photosynthesis to immunity to promote infection.

As one of the polyphagous and devastating phytopathogens, S. sclerotiorum causes Sclerotinia stem rot of soybean and rapeseed²⁹. A two-phase infection model based on cytological and genetic investigations has been widely accepted³⁰. For successful infection and colonization, S. sclerotiorum needs to secrete oxalic acid (OA) and effectors to counteract plant immunity and ensure pathogen success^31,32,33,34. Although the S. sclerotiorum genome encodes dozens (>70) of putative effectors, there are few studies on the molecular mechanism of their interaction with the host^{26,35,36,37,38}. Here, we identified a chorismate mutase (Cm) effector SsCm1 by screening virulence-related secreted proteins (SsVSPs). Unlike the known Cm effectors³⁹, SsCm1 shows no enzymatic activity and cannot interact with plant Cm. Functional genetic analyses and heterologous expression in plants confirmed that SsCm1, as an essential virulence factor, can inhibit basic immunity of plants and promote infection. Research on the target of effectors in hosts can serve as an important tool in identifying components of plant immune systems. We defined the conserved chloroplast RNA editing factor MORF2 as the functional target of SsCm1. Genetic analyses showed that MORF2 negatively regulates basic immunity and resistance to pathogens. Furthermore, biochemical evidences demonstrated that MORF2 is degraded during immunity stimulation, while SsCm1 stabilizes MORF2. We further showed that overexpression of SsCm1 or MORF2 in plants perturbed production of ROS, photoinhibition, and cell death. Overall, our results reveal that plants fine-tune the switch of chloroplast between photosynthesis and immunity by manipulating MORF2, and S. sclerotiorum secretes an enzymatically nonfunctional effector to promote infection via stabilizing MORF2.

Results

SsCm1 is an important effector secreted by S. sclerotiorum

A number of putative secreted proteins are encoded by S. sclerotiorum, and many of them are up-regulated during infection. Here, we defined them as SsVSPs^35,40,41. However, the functions of only a few SsVSPs have been characterized. By transiently expressing full-length SsVSPs^FL or signal peptide (SP)-removed SsVSPs^ΔSP in Nicotiana benthamiana, we found that SsVSP31^ΔSP promoted S. sclerotiorum infection (Fig. 1a). Due to the presence of a Cm domain (Supplementary Fig. 1a), SsVSP31 was renamed SsCm1. The expression of SsCm1 was continuously up-regulated in the early stage of infection in multiple hosts (Fig. 1b). The yeast invertase secretion assay confirmed that the SP of SsCm1 was functional and the specific detection of SsCm1-GFP in the culture supernatant confirmed that SsCm1 was secreted by S. sclerotiorum (Fig. 1c, d).

To explore the function of SsCm1 in S. sclerotiorum, two knockout mutants (ΔSsCm1-17 and ΔSsCm1-19) were obtained in the background of UF-1, and SsCm1-GFP was introduced into the ΔSsCm1-17 as a genetically complemented strain (ΔSsCm1-17-C32) (Supplementary Fig. 1). The pathogenicity of the ΔSsCm1 mutants on different hosts was significantly reduced compared with the UF-1 and the complemented strain, whereas no significant variations among these strains were detected in colony morphology, hyphal growth rate, compound appressorium formation, sclerotium development, OA production, and content of cell wall hydrolase (Fig. 1e and Supplementary Figs. 2 and 3). Similarly, deleting SsCm1 in the oxalate minus mutant background (Δoah)⁴² further reduced virulence (Supplementary Fig. 4). These data demonstrate that SsCm1 is secreted by S. sclerotiorum and plays an important role in the virulence of S. sclerotiorum.

SsCm1 lowers plant basic immunity and resistance to pathogens

To investigate the function of SsCm1 in plants, we first evaluated the effects of SsCm1 on several immune responses of N. benthamiana. The results showed that the transient expression of SsCm1 inhibited callose deposition and the expression of pathogenesis-related genes (NbPR1a and NbPR2) induced by PAMPs, including Flg22, chitin, and Sclerotinia culture filtrate elicitor (SCFE), while no significant variation was detected in the ROS burst induced by PAMPs (Supplementary Fig. 5). Together with SsCm1 promoting the virulence of S. sclerotiorum on N. benthamiana (Fig. 1a, e), suggesting that SsCm1 inhibits the basic immunity and resistance to S. sclerotiorum in N. benthamiana.

Next, stable 35S:SsCm1 overexpressing lines of Arabidopsis were created. Compared with wild-type Arabidopsis thaliana (Col-0) and GFP-expressing control lines, plants overexpressing SsCm1 showed significantly enhanced their sensitivity to UF-1, Botrytis cinerea, and the Δoah mutant. All plants showed normal growth and morphology (Fig. 2a, b). Similarly, SsCm1-expressing transgenic lines were more susceptible to the hemi-biotroph pathogen Pseudomonas syringae pv. tomato strain DC3000 (DC3000) (Fig. 2c, d). This demonstrates that SsCm1 inhibits resistance to a broad range of Arabidopsis pathogens.

Fig. 2: SsCm1 lowers the levels of basic immunity and resistance to pathogens in *Arabidopsis.*

Similarly, we also evaluated the effect of SsCm1-expression on the basic immunity processes in Arabidopsis. The results showed that compared with Col-0, 35S:SsCm1 overexpressing lines exhibited significantly decreased levels of callose deposition, stomatal closing, ethylene production, and the expression of immune marker genes, such as Flg22-induced receptor-like kinase 1 (FRK1), PR1, PR4, and phytoalexin deficient 3 (PAD3), following treatment with PAMPs (Fig. 2e–l). Furthermore, the expression of genes related to SA biosynthesis (ICS1 and SARD1) and the total SA content induced by PAMPs were also repressed in transgenic lines (Fig. 2m–o). Together, SsCm1 inhibits the basic immunity and resistance to pathogens in plants.

Enzymatically nonfunctional SsCm1 conservatively targets chloroplast RNA editing factors MORF2s

SsCm1 inhibits SA biosynthesis reminiscent of Cmu1 of Ustilago maydis which inhibits SA biosynthesis by interacting with the host Cm³⁹. Here, we showed that although there was weak interaction between SsCm1 and three Cms of soybean based on yeast two-hybrid (Y2H) experiments, these interactions were not reproduced in split-luciferase, bimolecular fluorescence complementarity (BiFC), and co-immunoprecipitation (Co-IP) assays in planta, and SsCm1 could form a self-dimer (Supplementary Fig. 6). Importantly, the heterologous expression of SsCm1 into yeast aro7 mutant could not restore the ability of yeast to synthesize tyrosine and phenylalanine (Fig. 3a), and the purified SsCm1^ΔSP showed lower Cm activity than that of UmCmu1 (Fig. 3b and Supplementary Fig. 7a). SsCm1 mixed with GmCms could not improve the enzyme activities in the reaction solutions (Fig. 3c and Supplementary Fig. 7a). Accordingly, the protein sequence of SsCm1 lacks a number of conserved amino acids reported in Cm and the amino acids key to Cm activity (Supplementary Fig. 7b)^39,43. Protein structure comparison showed that SsCm1 was obviously different from other Cms in three-dimensional structure, and molecular docking simulation revealed that SsCm1 showed less affinity with chorismate (binding energy = −2.1 kcal/mol) than ScAro7 (−5.7 kcal/mol) (Supplementary Fig. 7c, d). Another gene (SsCm2) of S. sclerotiorum encoded Cm, which was predicted to contain no SP, and the ability of SsCm2 to complement Scaro7 mutant and its enzymatic activity in vitro was equivalent to that of UmCmu1 (Fig. 3a, b and Supplementary Fig. 7). These results suggest that SsCm1 might acted in a different way than UmCmu1 and did not have functional chorismate mutase activity.

**Fig. 3: SsCm2 but not SsCm1 has chorismate mutase activity.**

To identify potential plant targets of SsCm1, we used SsCm1 as a bait to screen a soybean cDNA library and identified 11 SsCm1-interacting protein candidates (Fig. 4a). Interaction with Multiple Organellar RNA Editing Factor 2 (GmMORF2a) was detected with the highest frequency and selected for further studies. The interaction between full-length GmMORF2a and SsCm1 was confirmed by Y2H (Fig. 4b). Subcellular localization experiments showed that SsCm1, when expressed alone, was localized to the cytoplasm, nucleus, and a few chloroplasts, but when SsCm1 and GmMORF2a were co-expressed, SsCm1 was co-localized with GmMORF2a within chloroplasts (Fig. 4c). Subsequent BiFC and Co-IP assays confirmed that SsCm1 interacted with GmMORF2a in chloroplasts (Fig. 4d, e).

**Fig. 4: SsCm1 conservatively targets chloroplast RNA editing factors MORF2s.**

MORF2s are highly conserved chloroplast proteins encoded by nuclear genome⁴⁴. There are two copies of MORF2s in soybean (GmMORF2a/b), three copies in N. benthamiana (NbMORF2a/b/c) and only one AtMORF2 in A. thaliana. Here, we confirmed that these MORF2s were localized in chloroplasts (Supplementary Fig. 8). Furthermore, the interaction between SsCm1 and MORF2s were confirmed in planta through split-luciferase and Co-IP assays (Fig. 4f–h). Therefore, SsCm1 conservatively targets MORF2s. To explore whether SsCm1 can also target the host MORF2s when S. sclerotiorum naturally infects, we inoculated the strains expressing SsCm1-GFP on wild-type N. benthamiana, expressing GmMORF2a, and silencing NbMORF2s, respectively. The results showed that SsCm1 was secreted by S. sclerotiorum into plant cells around the infection sites and targeted to chloroplasts in a manner that depends on the content of host MORF2s (Supplementary Fig. 9), suggesting that SsCm1 can target chloroplasts facilitated by host MORF2s.

MORF2s negatively regulates basic immunity and resistance to pathogens

To investigate the role of MORF2 in plant immunity, we silenced all three NbMORF2s genes in N. benthamiana by virus-induced gene silencing (VIGS). The results showed that specific silencing of NbMORF2s resulted in a mild mosaic phenotype (Supplementary Fig. 10a, b). Importantly, silencing NbMORF2s significantly enhanced the resistance of N. benthamiana to UF-1 and B. cinerea, whereas the transient expression of AtMORF2, GmMORF2a, NbMORF2a, and NbMORF2b reduced the resistance (Supplementary Fig. 10c–g). These findings suggest that MORF2s negatively regulates resistance to pathogens in N. benthamiana.

Subsequently, stable 35S:GmMORF2a Arabidopsis overexpressing lines were created. Compared with Col-0, the GmMORF2a-overexpressing lines exhibited normal growth and morphology. In contrast, the knockout mutants of AtMORF2 were either unable to grow in the soil or able to grow slowly with small rosette leaves, consistent with the report that AtMORF2 regulates chloroplast development⁴⁴ (Fig. 5a). Importantly, the GmMORF2a-overexpressing lines were significantly more susceptible to UF-1, B. cinerea, Δoah mutant, and DC3000, whereas the morf2 mutants were significantly more resistant (Fig. 5a–d). Moreover, compared with Col-0, the GmMORF2a-overexpressing lines showed significantly lower levels in callose deposition, stomatal closing, ethylene production, accumulation of SA, and expression of basic immune marker genes and SA biosynthesis genes induced by PAMPs. Correspondingly, the morf2 mutants exhibited the characteristics of enhancing these immune responses, even in the absence of PAMP induction, suggesting a weak constitutive defense response (Fig. 5e–o). Therefore, our results demonstrate that MORF2 is negatively associated with basic immunity and resistance to pathogens in Arabidopsis.

Fig. 5: MORF2s attenuate basic immunity and resistance to pathogens in *Arabidopsis.*

Given the negative effects of MORF2s on plant immunity in N. benthamiana and A. thaliana, we also generated GmMORF2a and GmMORF2b loss-of-function mutants using CRISPR/Cas9 technology at two different targets. DNA sequencing identified two putative null mutants, Gmmorf2a/b-1 and Gmmorf2a/b-2, which harbored non multiple of 3-bp deletions in the first exon of GmMORF2a and GmMORF2b, causing frameshifts and premature termination of protein translation (Supplementary Fig. 11a). Morphological analysis showed that the Gmmorf2a/b mutants displayed dwarfish plants and spontaneous lesion mimic phenotypes on leaves (Supplementary Fig. 11b). Spontaneous lesion mimic mutants are usually accompanied by spontaneous cell death without pathogen infection and enhanced disease resistance⁴⁵. Consistent with this, the Gmmorf2a/b mutants significantly enhanced resistance to UF-1 (Supplementary Fig. 11c, d). The results suggest that MORF2s also negatively regulates resistance to S. sclerotiorum in soybean.

To investigate the functional relevance of SsCm1 and MORF2, NbMORF2s-silenced N. benthamiana, Arabidopsis morf2 mutants, and soybean Gmmorf2a/b mutants were used in inoculation assays. In NbMORF2s-silenced plants, the ΔSsCm1-17 mutant no longer exhibited weakened virulence compared with the UF-1, and the transient expression of SsCm1 caused no enhanced sensitivity of N. benthamiana to UF-1 (Supplementary Fig. 12a–d). Similarly, the ΔSsCm1-17 mutant exhibited no reduced virulence compared with the UF-1 in the Arabidopsis morf2 mutants or soybean Gmmorf2a/b mutants (Supplementary Figs. 11c, d and 12e, f). These results indicate that SsCm1 depends on MORF2s to promote virulence in plants. Taken together, SsCm1 promotes infection by targeting the chloroplast development related protein MORF2s, which functions as a negative regulatory factor for basic immunity and resistance to pathogens.

SsCm1 stabilizes MORF2s from degradation during immune activation

AtMORF2 was down-regulated at transcriptional level during avirulent effector AvrRps4-activated ETI and MPK3/MPK6 were constitutively activated^2,15. Reminiscent of the negative regulatory role of MORF2s in immunity, we surmise that MORF2s function is actively abolished during immune activation. In support of this hypothesis, the transcript of AtMORF2 was significantly down-regulated in response to PAMPs and pathogens infection (Supplementary Fig. 13a, b). Similarly, the transcript of NbMORF2s was significantly decreased following treatment with SCFE and UF-1 infection. Additionally, the transcript of GmMORF2a was also decreased after inoculation with UF-1 in soybean (Supplementary Fig. 13c, d). These results indicate that MORF2s are down-regulated at the transcriptional level during immune activation.

Interestingly, the co-expression of GmMORF2a-GFP and SsCm1-RFP showed stronger GFP fluorescence signal than that of GmMORF2a-GFP expressed alone (Fig. 6a, b). To more directly examine the effect of SsCm1 on MORF2s stability, we compared the accumulation of six MORF2s-GFP proteins in N. benthamiana co-expressed with SsCm1 or SsVSP25, another S. sclerotiorum effector that also targets chloroplasts but not interacting with GmMORF2a-GFP. The results showed that the six MORF2s-GFP proteins accumulated to much higher levels in the presence of SsCm1 than that of the co-expression with SsVSP25, while GmPETE, another SsCm1-interacting protein, accumulated at the similar level when co-expressed with SsCm1 or SsVSP25 (Fig. 6c). Furthermore, the cell-free degradation assays also confirmed that MORF2s would be degraded and SsCm1 could alleviate the degradation (Fig. 6d). There results suggest that MORF2s are unstable, while SsCm1 can specifically stabilize the MORF2s to promote their accumulation.

**Fig. 6: MORF2s are degraded during immune activation and stabilized by SsCm1.**

Previous study showed that AtMORF2 protein was reduced by about 90% in senescent leaves⁴⁶. To explore the stability of the GmMORF2a protein during immune activation, we treated N. benthamiana leaves transiently expressing GmMORF2a-GFP with the three types of PAMPs (Flg22, Chitin, and SCFE) and three different pathogens (B. cinerea, ΔSsCm1-17 mutant, and DC3000). The results showed that, compared to the control treatment, the GmMORF2a-GFP protein degraded rapidly after PAMPs or pathogens treatments, particularly during the ΔSsCm1-17 treatment. Importantly, co-expression of SsCm1 significantly weakened the degradation of the GmMORF2a-GFP induced by PAMPs or pathogens treatments (Fig. 6e). Likewise, the other five MORF2s proteins were rapidly degraded after SCFE treatment, and NbMORF2a and AtMORF2 were also degraded following induction by Flg22, whereas the GmPETE protein level was not affected by SCFE treatment (Fig. 6f). These results indicate that MORF2s are rapidly degraded during immune activation, and SsCm1 can inhibit this degradation.

To investigate the stability of endogenous MORF2 proteins during immune activation, we synthesized the specific antibody of MORF2 and confirmed its applicability (Supplementary Fig. 14). Short-term treatment of A. thaliana Col-0 with SCFE of S. sclerotiorum UF-1 caused a slight decrease in the amount of endogenous MORF2 protein, while treatment with SCFE of the ΔSsCm1-17 mutant caused a more significant decrease. Moreover, UF-1 inoculation also resulted in less degradation of endogenous MORF2 protein than that of the ΔSsCm1-17 mutant inoculation (Fig. 6g). Likewise, the detection of endogenous MORF2s protein in soybean showed similar results (Fig. 6h). To confirm that SsCm1 secreted by S. sclerotiorum could reduce the degradation of MORF2s in chloroplasts, we incubated the extracted chloroplastic proteins with SCFE from UF-1 and the ΔSsCm1-17 mutant, respectively. The results of western blot revealed that compared with SCFE of the ΔSsCm1-17 mutant, SCFE of UF-1 could retain higher MORF2s protein level in chloroplasts, indicating that SsCm1 secreted by UF-1 could also inhibit the degradation of MORF2s in chloroplasts, similar to purified SsCm1, although the inhibition effect was not as strong as that of the purified SsCm1 (Fig. 6i). The fluorescence results also exhibited that inoculation with S. sclerotiorum could significantly reduce the content of GmMORF2a-GFP in chloroplasts, while SsCm1 secreted by UF-1 could significantly inhibit this reduction (Fig. 6j, k). These results suggest that endogenous MORF2s in plants can also be degraded after immune stimulation, and the physiological level of SsCm1 secreted by S. sclerotiorum in the early stage of infection can inhibit the degradation of MORF2s in chloroplasts to some extent.

SsCm1 and MORF2s inhibit the accumulation of ROS and cell death

When evaluating the effects of SsCm1 and GmMORF2a on various immune outputs, we found that the overexpression of SsCm1 and GmMORF2a not only widely inhibited basic immunity, but also inhibited N. benthamiana cell death triggered by different cell death inducing factors, including the constitutively active variant of AtMKK5 (AtMKK5^DD, abbreviated as DD)⁴⁷, the necrosis-inducing effector of S. sclerotiorum (SsNE2)⁴⁸, and the Phytophthora elicitor (INF1)⁴⁹, whereas SsCm1 and GmMORF2a could not induce cell death by themselves (Supplementary Fig. 15a). We observed that overexpression of GmMORF2a could significantly alleviate the level of cell death, while overexpression of SsCm1 slightly weakened cell death, whereas no significant difference in the score of hypersensitive response (HR) compared with GFP control (Supplementary Fig. 15b–d). Similarly, overexpression of NbMORF2a and AtMORF2 significantly alleviated cell death. Considering that SsCm1 targets MORF2s in chloroplasts, we generated a chimera cTP-SsCm1 (chloroplast targeting peptide of NbMORF2b added to the N-terminal of SsCm1-GFP) (Supplementary Fig. 16). Overexpression of cTP-SsCm1 significantly inhibited cell death, comparable to the overexpression of MORF2s. Importantly, after silencing NbMORF2s, cTP-SsCm1 lost its ability to inhibit cell death (Supplementary Fig. 15a–d). These results suggest that MORF2s is a general suppressor of cell death, and SsCm1 inhibits cell death by targeting MORF2s in the chloroplast. Furthermore, 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining of leaves prior to visible cell death also confirmed that overexpression of MORF2s or SsCm1 inhibited the accumulation of ROS before triggering cell death. It is worth noting that SsCm1-GFP can effectively inhibit the accumulation of ROS, suggesting that SsCm1 in planta can reduce the accumulation of ROS and delay the subsequent cell death (Supplementary Fig. 15e, f).

To evaluate the effects of SsCm1 and MORF2s on cell death in A. thaliana, dexamethasone-induced expression of DD in Arabidopsis transgenic plants leading to constitutive activation of MAPK3/6⁵⁰ were used to hybridize with morf2 mutant lines, GmMORF2a-overexpressing lines, and SsCm1-overexpressing lines, respectively. We found that overexpression of GmMORF2a or SsCm1 in DD background (DD/GmMORF2a or DD/SsCm1) inhibited cell death and ion leakage after dexamethasone treatment, and inhibited ROS accumulation prior to visible cell death (Fig. 7a–c). Moreover, up-regulated expression of defense genes PR1, PR2, and PAD4 caused by constitutive activation of MAPK3/6 was also weakened in DD/GmMORF2a and DD/SsCm1 plants, but was enhanced in DD/morf2-1 plants (Fig. 7d). These results suggest that overexpression of GmMORF2a or SsCm1 inhibits ROS accumulation and cell death induced by MAPK3/6 constitutive activation.

Fig. 7: SsCm1 and GmMORF2a inhibit ROS accumulation and cell death in *Arabidopsis.*

Chloroplast ROS accumulation and cell death is necessary for ETI and robust plant immunity¹⁵. Therefore, we evaluated the levels of ROS and cell death during infiltration with DC3000 carrying AvrRpt2 (Pst-AvrRpt2), a DC3000 strain carrying the avirulence effector (AvrRpt2) recognized by RPS2. Here, cell death, ion leakage, and ROS accumulation induced by high titer Pst-AvrRpt2 inoculation were weakened in both GmMORF2a-overexpressing lines and SsCm1-overexpressing lines (Fig. 7e–g). Similarly, overexpression of GmMORF2a or SsCm1 also alleviated ROS accumulation upon UF-1 treatment (Fig. 7g). These results suggest that MORF2 and SsCm1 negatively regulate the accumulation of ROS and cell death during pathogens infection.

We further investigated the role of SsCm1 in inhibiting plant cell death and ROS accumulation during the early infection of S. sclerotiorum. The inoculation experiments were carried out with weakly virulent strains Δoah and Δoah/ΔSsCm1-9 to avoid the strong pathogenicity of the UF-1. By measuring the ion leakage in the inoculation areas, it was found that strain Δoah caused significantly less ion leakage than strain Δoah/ΔSsCm1-9 (Fig. 7h). Consistently, in the early stage of infection, strain Δoah also caused less ROS accumulation than strain Δoah/ΔSsCm1-9 (Fig. 7i). These findings confirmed that S. sclerotiorum effectively suppressed host cell death and ROS accumulation through secreting SsCm1 in the early infection.

SsCm1 and MORF2s alleviate photosynthetic inhibition

MORF2s and SsCm1 inhibit the accumulation of ROS and cell death caused by MAPK3/6 constitutive activation, and MORF2s, as chloroplast protein, participates in chloroplast development and the maturation of RNA encoding photosynthetic elements⁴⁴, which reminds us that MORF2s and SsCm1 may play roles in maintaining the efficient operation of photosynthetic apparatus to alleviate photosynthetic inhibition, which is necessary for ROS accumulation and cell death¹⁵. In support of this hypothesis, upon MPK3/MPK6 activation, the decrease of maximal PSII activity parameter Fv/Fm is significantly weakened in DD/GmMORF2a and DD/SsCm1 plants, and is further reduced in DD/morf2-1 plants (Fig. 8a). Additionally, the dramatic inhibition of photosynthesis related genes LHCB1.2, PETE, and PsbA after MPK3/MPK6 activation was also significantly alleviated in DD/GmMORF2a and DD/SsCm1 plants (Fig. 8b). Consistent with the finding that overexpression of GmMORF2a or SsCm1 inhibited the accumulation of ROS and cell death induced by Pst-AvrRpt2 inoculation, overexpression of GmMORF2a or SsCm1 also alleviated the decrease of Fv/Fm and the down-regulation of photosynthesis-related genes induced by Pst-AvrRpt2 inoculation, which is necessary for HR cell death in plant immunity¹⁵ (Fig. 8c–e). Importantly, inoculation with strain Δoah of S. sclerotiorum showed a slower rate of Fv/Fm decline than inoculation with strain Δoah/ΔSsCm1-9 (Fig. 8f), suggesting that physiological level of SsCm1 secreted by S. sclerotiorum could delay the photosynthetic inhibition caused by S. sclerotiorum infection. Similarly, SCFE treatment, as well as UF-1 and DC3000 inoculation also induced the decrease of Fv/Fm and the down-regulation of photosynthesis-related genes²⁷, while overexpression of GmMORF2a or SsCm1 significantly alleviated these decreases, which were enhanced in morf2 mutants (Fig. 8g–j and Supplementary Fig. 17). These results suggest that GmMORF2a and SsCm1 alleviate immunization induced photosynthetic inhibition, which may lead to the inhibition of ROS accumulation and cell death.

**Fig. 8: Overexpression of SsCm1 or GmMORF2a alleviates photosynthetic inhibition under immunization.**

Discussion

To cope with inevitable abiotic stresses and numerous invaders, plants have evolved mechanisms that sacrifice growth by consuming energy to build defensive barriers, adapt to environmental change, and eliminate invaders^51,52. Chloroplast, as an integrator that perceives the environment, needs to dynamically switch the photosynthetic apparatus on and off to achieve a proper growth-defense tradeoff⁹. Firstly, chloroplasts actively down-regulate⁵³ and reduce the processing of RNA encoding genes in photosynthesis-related apparatus⁵⁴, control the routes of chloroplast protein import⁵⁵, phosphorylate chloroplast proteins to inhibit electron transport rate^56,57, reduce cyclic electron flow (CEF), reduce NPQ, and degrade PSII^15,58, and finally inhibit photosynthesis to reduce energy obtained by other cellular activities and pathogens and prioritize energy investments. Secondly, when the threats are lethal, a robust defense, but not growth, is of the highest priority. Chloroplasts are also the main source of ROS. They actively disrupt the stoichiometry of photosynthetic proteins, degrade PSII, and change the redox state of the plastoquinone pool, resulting in the excessive production and accumulation of ROS, which eventually triggers a cell death that resembles the effector-triggered HR that limits the growth of pathogens^{17,23,24,59,60,61}. Thirdly, ¹O₂ and tetrapyrrole are used as RS molecules to drive transcriptional reprogramming, which increases SA biosynthesis and nuclear-encoded defense gene expression, resulting in extensive basic immune output and ROS burst^{6,7,17,23,24,60}. However, there are still confused issues about how chloroplasts can quickly and accurately achieve this transition from growth to defense and which components are responsible for this transition. Here, we found that plants fine-tune chloroplasts transitioning from photosynthetic state to immune state by reducing the level of MORF2s. Overexpression of MORF2 alleviates the immunization-induced photosynthetic inhibition and down-regulation of photosynthesis related genes and inhibits the accumulation of ROS and the subsequent cell death. In the meantime, MORF2 overexpression inhibits the output of various basic immunity and SA biosynthesis, leading to a weakened resistance to pathogens, while the corresponding loss of MORF2 shows the opposite phenomenon. In addition, the rapid degradation of MORF2s proteins and down-regulation of MORF2s expression during immune activation emphasizes that plants can spontaneously turn on the immune activation of chloroplasts. Importantly, S. sclerotiorum has evolved a strategy to subvert this process by secreting effector SsCm1 to stabilize MORF2s, effectively maintaining chloroplasts in a growth state and thereby facilitating infection.

AtMORF2 was originally characterized as an RNA editing factor of chloroplasts⁴⁴, and the mitochondrial NbMORF8 was implicated as a negative regulator of immunity through the regulation of ROS accumulation and SA signaling⁶². OVEREXPRESSOR OF CATIONIC PEROXIDASE3 negatively regulates immunity and fungal resistance through controlling chloroplast NADH dehydrogenase (ndh) transcript editing⁵⁴. Here, we find that MORF2s also negatively regulate immunity, possibly by mediating RNA editing of certain chloroplast proteins. Moreover, AtMORF2 may also be implicated in RS. AtMORF2 acts as chaperone that directly promotes the accumulation of several tetrapyrrole biosynthetic enzymes and participates in tetrapyrrole biosynthesis (TBS)^46,63,64. This is reminiscent of PROGRAMMED CELL DEATH8, which negatively regulates RS, ROS accumulation and cell death by interacting with TBS enzymes⁶⁵. RS promotes the expression of nuclear-encoded defense genes and SA synthesis¹². AtMORF2 also directly interacts with GUN1 to participate in RS⁶⁶ and several genome uncoupled (gun) mutants are susceptible to pathogens⁶. Consistent with the involvement of MORF2 in RS⁶⁶, our results showed that overexpression of GmMORF2a or SsCm1 alleviates the down-regulation of immunization-induced RS marker gene LHCB1.2 (Fig. 8b, d, e, i, j). Therefore, we speculate that MORF2s inhibited immunization-induced RS by affecting TBS or directly interacting with GUN1, which could lead to the alleviation of photosynthetic inhibition, ROS accumulation, SA biosynthesis, basic immune output, and cell death, while SsCm1 inhibited RS by stabilizing MORF2s. Further research is needed to explore how MORF2s inhibits RS and how RS affects immunity.

Consistent with MORF2s functioning as negative regulators of plant immunity, the expression of MORF2s is suppressed during immune activation, which is similar to OCP3 and chloroplast RNA editor CRR21^54,67, emphasizing that plants actively down-regulate immune negative regulators as a defense strategy. Similarly, stresses also regulate the degradation and input of chloroplast proteins through ubiquitination, protease, autophagy, senescence-associated vacuoles, and chloroplast vesiculation^{55,56,68,69,70,71,72,73}, and the instability of AtMORF2 protein has been reported^46,64. Here, we found that MORF2s were degraded within 30 min following immune activation, suggesting that plants directly activate unknown degradation systems to degrade MORF2s after sensing pathogen signals. Consistently, the pathogen-triggered rapid editing inhibition in ndh transcripts leads to NDH destabilization and subsequent inhibition of CEF⁵⁴. This suggests that the rapid degradation of chloroplast editing factors may be ubiquitous, as such, the degradation of MORF2s may promote the inhibition of extensive RNA editing, thereby transitioning chloroplasts from the photosynthetic state to the immune state. These findings are consistent with the recent discovery, showing that MORF8 forms condensates under heat stress and recruits RNA editor elements, such as MORF2, to inhibit RNA editing and reduce photosynthesis⁷⁴, implicating a common phenomenon, i.e., the reduction of the function of MORFs, such as the degradation of MORF2 and the formation of condensates of MORF8, decreases the level of photosynthesis under stress. To further reveal the key factors responsible for MORF2s degradation and the mechanism of immune signal-induced MORF2s degradation will provide new insights for understanding how chloroplasts balance photosynthesis and defense.

Correspondingly, S. sclerotiorum evolved effector SsCm1 to promote infection by directly targeting and stabilizing MORF2s to prevent chloroplasts from entering immune state. This is reflected in the phenotypic similarity between overexpressed SsCm1 and overexpressed GmMORF2a, which both alleviated immune-induced photoinhibition and inhibited ROS accumulation, cell death, basic immunity and pathogen resistance. In addition, the physiological level of SsCm1 secreted by S. sclerotiorum could also alleviate photoinhibition and inhibit ROS accumulation and cell death during infection. Crucially, the promotion of infection by SsCm1 depends entirely on MORF2s, and the virulence function of SsCm1 is lost after silencing or losing MORF2s, which further emphasizes that MORF2s are the physiological targets of SsCm1. Notably, based on the analysis of conserved domains, SsCm1 has always been regarded as the homologs of U. maydis effector Cmu1^34,35,40, a classical effector that relies on the activity of Cm and the interaction with ZmCm2, and in turn mediates metabolic priming, especially lowering the available substrate for SA biosynthesis³⁹. However, we demonstrated in yeast complementation experiments and in vitro enzyme activity experiments that SsCm1 could not restore the growth of yeast aro7 mutant like Cmu1, nor did it have enzyme activity, indicating that SsCm1 is an enzymatically nonfunctional effector and has adopted different strategies to suppress plant immunity. This is different from the reported Cm effectors, which all have enzymatic activity^39,75,76. A careful sequence alignment of SsCm1 and other Cms showed that SsCm1 has obvious differentiation in several reported essential amino acids for enzyme activity, such as A93 and V149 of SsCm1 are K and T respectively in the Cms with enzyme function, and also showed differences in many conserved amino acids^39,43. The AlphaFold prediction also showed that the three-dimensional structure of SsCm1 is obviously different from the Cms with enzyme function, suggesting that the structural variation caused by the differentiation of key amino acids in enzyme activity may be the reason why SsCm1 loses its enzyme function. Notably, all known enzymatic functional Cm effectors are derived from biotrophic organisms^39,75,76, which is different from SsCm1 from S. sclerotiorum, a necrotrophic or hemi-necrotrophic pathogens. Interestingly, the hypothesized Cm effectors are rare in necrotrophs and saprophytes, and is only identified in S. sclerotiorum³⁹. We speculate that there are two possibilities for this difference in distribution of Cm effectors: (1) Metabolic priming by Cm effectors may lead to adverse reactions that are not conducive to necrotrophs, such as the accumulation of some harmful metabolites^39,77 or the lack of preferred metabolites; (2) There is no urgent need for Cm effectors to inhibit certain specified plant defense responses in the pathogenic process of necrotrophs⁷⁸, so the evolutionary selection pressure does not force necrotrophs to evolve enzymatic functional Cm effectors. Further detailed and comprehensive analysis of the evolution of Cm effectors will probably reveal the mystery. We noticed that SsCm1 does not contain the recognizable cTP, but it can be partially localized in chloroplasts, and artificially expressing SsCm1 in chloroplasts can more effectively inhibit elicitor-induced ROS accumulation and cell death, indicating that SsCm1 mainly functions in chloroplasts. This raises a question, how does SsCm1 target chloroplasts? Considering SsCm1 significantly increased chloroplast localization when co-expressed with MORF2, we speculate that SsCm1 may be migrated to chloroplast through interaction with MORF2. In addition, we cannot rule out the possibility that SsCm1 may enter chloroplasts through non-classical input mechanism or interaction with other chloroplast proteins⁶, and the explicit mechanism underlying the transportion of SsCm1 into chloroplasts needs further study.

Overall, we propose that when plants sense PAMPs or pathogens, they trigger unknown degradation systems to rapidly degrade MORF2s, which damages the RNA editing process of chloroplast proteins and TBS pathway, leading to photosystem damage, photosynthesis inhibition, ROS accumulation, triggering of RS, and transition of chloroplast from growth state to immune state. The RS is transmitted to the nucleus to promote the up-regulation of defense genes, such as SA biosynthesis genes, and down-regulation of PhANGs, ultimately leading to the synthesis of SA and the output of basic immunity, further strengthening the active inhibition of photosynthesis and the accumulation of ROS, and finally causing cell death and disease resistance (Fig. 9a). Correspondingly, the secreted enzymatically nonfunctional effector SsCm1 of S. sclerotiorum directly targets and stabilizes MORF2s, thus maintaining the photosynthetic state of chloroplasts, which reduces resistance of plants to pathogens (Fig. 9b).

**Fig. 9: Proposed model for SsCm1 promoting infection via stabilizing MORF2s, a chloroplast growth-defense switch.**

Methods

Plant materials and growth conditions

Nicotiana benthamiana wild-type and gene silencing lines, Phaseolus vulgaris, Solanum lycopersicum, and soybean wild-type (Glycine max cv. Williams 82) and the Gmmorf2a/b mutants were maintained at 22 °C in a growth chamber with a 16 h day/8 h night cycle (100–150 μmol photons m⁻² s⁻¹).

All Arabidopsis plants used in this study were in Col-0 background. Arabidopsis mutant seeds of morf2-1 (SALK_094930) and morf2-1 (SALK_149307) were obtained from the Nottingham Arabidopsis Stock Centre. The dexamethasone-inducible promoter-driven AtMKK5^DD transgenic plants (DD) were reported previously⁵⁰. Soil-grown Arabidopsis plants were grown in a growth chamber at 22 °C and approximately 70% relative humidity with a 10 h day/14 h night cycle (100–150 μmol photons m⁻² s⁻¹). For experiments related to cell death and photosynthesis, the treated Arabidopsis plants were kept for the times indicated in the corresponding experiments under continuous light (100 μmol photons m⁻² s⁻¹).

Pathogen strains and inoculation assays

The wild-type strain UF-1 and its derivative strains of S. sclerotiorum were initially grown on Potato Dextrose Agar (PDA) plates cultured at 25 °C. The oxalic acid minus mutant (Δoah 3-6-1) of S. sclerotiorum was reported previously⁴². Mycelium plugs or pure fresh mycelia were collected from fresh mycelia growing on PDA plates or cultured in Potato Dextrose Broth (PDB) for 2 d, inoculated onto plant leaves, and maintained at high humidity.

The B. cinerea strain B05.10 was used in this study. Mycelium plugs (0.5 cm in diameter) or 2 × 10⁶/mL spore suspension with 0.01% Tween 20 were used to inoculate plant leaves and maintained at high humidity.

Pseudomonas syringae pv. tomato strain DC3000 containing an empty vector (DC3000) or AvrRpt2 (Pst-AvrRpt2) was cultured overnight at 28 °C in LB medium containing 25 μg/mL rifampicin or 25 μg/mL rifampicin with 50 μg/mL kanamycin. For disease evaluation caused by DC3000, the DC3000 strain was resuspended in 10 mM MgCl₂ at OD₆₀₀ = 0.0005. The bacterial suspensions were then infiltrated into 4-to-5-week-old Arabidopsis leaves. The numbers of bacterial cells were determined by colony-forming units per cm² (CFU/cm²) of leaf tissue at 3 d post-infiltration (dpi) as previously described⁵⁰. For the experiments related to photosynthesis, Arabidopsis leaves were infiltrated with DC3000 (OD₆₀₀ = 0.2) or Pst-AvrRpt2 (OD₆₀₀ = 0.2), and for the experiments related to cell death, Arabidopsis leaves were infiltrated with Pst-AvrRpt2 (OD₆₀₀ = 0.02).

Transient expression, protein extraction, and western blots analysis

Agrobacterium-mediated transient expression in N. benthamiana and protein extraction were performed as previously described⁵⁰. Before infiltration, the bacterial suspension carrying different constructs was adjusted to a final OD₆₀₀ = 0.6. For experiments that required co-infiltration, Agrobacterium suspensions carrying different constructs were mixed at a 1:1 ratio before infiltration. Samples were collected at 36 h post inoculation (hpi) or 48 hpi for analysis based on experimental requirements. For the expression of cell death inducing factors, Agrobacterium expressing AtMKK5^DD or INF1 was infiltrated with OD₆₀₀ = 0.2, and Agrobacterium expressing SsNE2 was infiltrated with OD₆₀₀ = 0.6.

Plant tissues were frozen and ground in liquid nitrogen and then extracted according to instructions of the Plant Protein Extraction Kit (CW0885, CWBIO) containing 1% (v/v) plant protease inhibitor cocktail (P9599, Sigma). For protein extraction of S. sclerotiorum, 0.2 g of fresh hyphae was used according to the Minute^TM Total Protein Extraction Kit for Microbes with Thick Cell Walls (YT-015, Invent). The supernatant of protein extracts was heated at 70 °C for 10 min in SDS loading buffer (2% (w/v) SDS, 10% (w/v) glycerol, 0.01% (w/v) bromophenol blue and 0.125 M Tris-HCl, 0.3 M DTT, pH 6.8) and loaded on SDS-PAGE acrylamide gels for western blotting. All immunoblots were analyzed using appropriate antibodies as indicated in the figures. Blots were stained with Coomassie Brilliant Blue (CBB) to verify equal loading. Uncropped blots are provided in Supplementary Fig. 18.

RNA isolation and quantitative RT-PCR

Samples for gene expression analysis were frozen and ground in liquid nitrogen and then total RNA purification, reverse transcription, and qRT-PCR were completed using one-step kit (TransGen Biotech). The S. sclerotiorum β-tubulin gene Sstublin, G. max actin gene GmActin, A. thaliana actin gene AtActin2, and N. benthamiana NbEF1α gene were used to normalize transcript levels. The relative expression levels were calculated with three technical replicates using the 2^−ΔΔCt method. All primers used for qRT-PCR were listed in Supplementary Data 1.

Secretion assays and production of SCFE

The predicted N-terminal 20-amino acid SP sequences of SsCm1 were fused in frame to the invertase gene in the pSUC2 vector by gene synthesis in GenScript Biotech. The construct containing the SP of effector Avr1b were used as positive controls as previously described⁷⁹. Recombinant constructs were transformed into yeast and SP secretion confirmation was performed using the Signal peptide secretion yeast detection kit (DZSL1561, Coolaber).

For the detection of SsCm1 secretion by S. sclerotiorum, the strains carrying GFP or SsCm1-GFP were inoculated into PDB in the presence of N. benthamiana leaf discs for 24 h. Culture medium was harvested by filtration through a 75-µm nylon mesh and the supernatant was concentrated by freeze-drying. Total proteins were extracted from mycelia or supernatant, and immunoblotted with rabbit polyclonal anti-GFP antibody (D110008, 1:5000; Sangon). To obtain SCFE, the UF-1 or the ΔSsCm1-17 mutant strain was inoculated into PDB in the presence of 10 µM 1,16-Hexadecanediol (S68032, Yuanye) for 24 h. Culture medium was harvested by filtration through a 75-µm nylon mesh and freeze-drying the concentrated supernatant. The dried material was resuspended in H₂O (0.5 g dry weight/mL) and centrifuged twice for 20 min at 10,000 × g and 4 °C to remove insoluble particles. SCFE activity was identified by measurement of ethylene production in A. thaliana leaves following treatment with the SCFE solution as previously described⁸⁰.

Genetic manipulation and developmental phenotype evaluation in S. sclerotiorum

Gene deletion mutants of the SsCm1 gene in isolate UF-1 and the Δoah strain of S. sclerotiorum were generated using a homologous recombination strategy using the protoplast transformation method⁸¹. Transformants were purified by hygromycin selection and were verified by PCR. Uncropped gels are provided in Supplementary Fig. 18. To genetically complement the SsCm1 knockout mutants, a SsCm1-GFP fusion complementation vector based on pYF11 was generated and was transformed into the ΔSsCm1-17 mutant. Transformants were purified using G418 selection and were verified by RT-PCR.

The evaluation of developmental phenotype of S. sclerotiorum was performed as previously described⁸¹. The strains were inoculated onto PDA plates for 2 d or 14 d at 25 °C and the dry weight of sclerotia was measured. Glass slides and onion epidermal tissue were used to induce the formation of infection cushions in vitro and in vivo, respectively. The strains cultured on PDA with 0.005% (w/v) bromophenol blue for 36 h were used to evaluate the production of OA. The enzymatic activities of cutinase and cellulase were measured by mycelium according to the previously described⁸² and the manufacturer’s instructions of the Cellulase test kit (A138, Nanjing Jiancheng), respectively.

Plasmid construction and generation of transgenic and gene silencing plants

To screen SsVSPs, the full-length coding region (SsVSPs^FL) or the sequence with the predicted SP removed and artificial start codon added at the N-terminal (SsVSPs^ΔSP) was cloned into the pCHF1301-3×FLAG vector. Unless otherwise specified, the SsCm1^ΔSP was used in this study. To generate the SsCm1-GFP and MORF2s-GFP constructs, the corresponding coding sequences were cloned into pCHF3301-GFP driven by the CaMV 35S promoter. All recombinant constructs were transformed into Agrobacterium GV3101 and then 35S:SsCm1 or 35S:GmMORF2a was transformed in Arabidopsis Col-0 through the floral dipping method. Transgenic Arabidopsis plants were selected using Basta (10 µg/mL). All experiments using transgenic Arabidopsis plants were performed using two independent T4 homozygous lines. The DD/morf2-1, DD/GmMORF2a, and DD/SsCm1 plants were created by crossing the homozygous plants, and homozygous lines were selected and confirmed by PCR.

VIGS assays were performed as previously described⁵⁰. Briefly, independent cultures of Agrobacterium carrying pTRV1 or pTRV2-based constructs were resuspended in 10 mM MgCl₂, 10 mM MES (pH 5.6) and 150 µM acetosyringone at OD₆₀₀ = 0.8, and incubated for 2 h at 28 °C in the dark. Cultures were mixed at a 1:1 ratio and this suspension was used to infiltrate the stem and underside of cotyledons of 2-week-old N. benthamiana seedlings.

To generate the CRISPR/Cas9-engineered Gmmorf2a/b mutant, gRNAs were designed using the CRISPR-P website (http://crispr.hzau.edu.cn). Two 20-bp DNA fragments coding for the gRNA were inserted into the pCas9-MDC-sgRNA plasmid. Plasmids were individually introduced into Agrobacterium strain EHA105 via electroporation and then transformed into WT soybean (Williams 82) using the cotyledon-node method⁸³. The mutant lines were identified by DNA sequencing. All oligonucleotide primers were listed in Supplementary Data 1.

PAMP treatments and determinations of basic immunity

Except for the luminol-based ROS burst assays, leaves were infiltrated with water (Mock), 1 µM flg22 (abs45152926, Absin), 200 µg/mL chitin (C9752, Sigma), or 50 µg/mL SCFE, and assayed for basic immunity or protein extractions at the designated times were indicated in the corresponding figures. The luminol-based ROS burst assays were performed as previously described⁵⁰. Briefly, N. benthamiana leaf discs transiently expressing the SsCm1-FLAG or GFP-FLAG (as control) were placed in white 96-well plates (655074, Greiner) with 200 µL water overnight. Then, water was replaced by a solution containing individual PAMPs (100 nM flg22, 50 µg/mL chitin, or 2 µg/mL SCFE), 100 µM luminol (123072, Sigma), and 20 µg/mL HRP (HY-125859, MCE), and luminescence was measured in a microplate reader (PerkinElmer) for 60 or 90 min. For data analysis, both relative luminescence units (RLU) produced every minute and total RLU were plotted.

Callose quantification was performed as previously described⁵. The leaves of Arabidopsis or N. benthamiana were infiltrated with PAMPs or water using a needleless syringe and covered for 24 h. Chlorophyll was removed by incubating in ethanol (90% v/v). The destained leaves were vacuum-infiltrated with 0.01% aniline blue (S19056, Yuanye) and incubated for 1 h. Then, aniline blue was removed and callose deposits were visualized under UV illumination (excitation, 365 nm; emission, 460 nm) and quantified using ImageJ.

Stomatal aperture measurements were performed as previously described⁷. Arabidopsis leaves were detached and floated in the incubation buffer (10 mM MES, 10 mM KCl, 50 μM CaCl₂) for 2 h at 22 °C with 100 μmol photons m⁻² s⁻¹ light. Then, leaves were treated with PAMPs or water and incubated for an additional 6 h. After the incubation, epidermal strips of the leaves were observed under microscopy. The stomatal aperture was calculated as the ratio of the pore width/guard cell length.

Ethylene and SA content were determined as previously described^7,80. Leaves were treated with water or PAMPs for 6 h and ethylene emission was measured by gas chromatography. Leaves were treated with water (Mock) or PAMPs for 12 h and total SA content was quantified by high-performance liquid chromatography.

For the determination of defense-related genes expression, leaves of Arabidopsis plants or N. benthamiana were infiltrated with PAMPs or water for 12 h and tissues were snap-frozen. The expression of genes was normalized to the levels of AtActin2 or NbEF1α transcripts and presented as fold induction compared with the expression before treatment which was set as the baseline of 1.

Y2H screening and confirmation

The Y2H assay was used to screen a soybean cDNA library to identify potential targets of SsCm1 and to confirm the specific interaction. The coding sequence of SsCm1 (without the SP, SsCm1^ΔSP) was cloned to the bait vector pGBKT7 and transformed into the Y2H Gold strain. The bait strain was mated with the Y187 yeast strain (harboring a soybean Mate & Plate Libraries), and the matings were selected on the synthetic medium (SD) without leucine, tryptophan, histidine, and adenine (SD-L/W/H/A) agar plates. The inserts in the prey vector were confirmed using yeast plasmid sequencing. The potential interactors were subjected to BLAST (http://www.ncbi.nlm.nih.gov/) analyses for identification and confirmation of the correct orientation of the insert sequences. The coding sequence of full-length potential interactors was cloned into the prey vector pGADT7 and co-transformed with pGBKT7-SsCm1^ΔSP into Y2HGold yeast cells. All transformations were plated on SD-L/W, SD-L/W/H + X-α-Gal (630463, Clontech), and SD-L/W/H/A + X-α-Gal agar plates. For the quantitative evaluation of the results of Y2H, the transformed single colony with the same size on SD-L/W medium was transferred to the 10 mL liquid SD-L/W/H/A medium, and the OD₆₀₀ values were determined after shaking culture for 24 h. The pGBKT7-53 with pGADT7-T was used as a positive control, and pGBKT7-Lam with pGADT7-T was used as a negative control.

Yeast complementation and determinations of chorismate mutase activity

The assays of gene functional complementation of ScARO7 in yeast were performed as previously described with modifications³⁹. Briefly, yeast strain Y054679 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YPR060c::kanMX4, Aro7, Euroscarf) was transformed with the corresponding pGADT7 derivatives using standard protocols (Clontech) and tested for growth on SD without leucine, phenylalanine, and tryptophan.

Chorismate mutase activity assays were performed as previously described⁸⁴. SsCm1^ΔSP-HA, SsCm2-HA, GmCms-HA, and UmCmu1^ΔSP-HA (positive control) were cloned into pGEX-6P-1 vector, respectively, and the proteins were obtained in Escherichia coli BL21 and purified by glutathione affinity purification (C600327, Sangon). Then, the GST moiety was removed by PreScission protease (C610303, Sangon) and chorismite mutase activity assays were performed. After acidic conversion of prephenate to phenylpyruvate, the reaction was basidified and the extinction at 320 nm was measured. The increase in extinction was plotted against time (in min) to visualize the formation of phenylpyruvate, and purified GST protein was used as a negative control.

Subcellular localization and BiFC assays

For subcellular localization, N. benthamiana leaves expressing GFP- or RFP-fused proteins at 2 dpi were imaged with a Leica confocal microscope (Leica Microsystems) using LAS-X software with the preset settings for GFP (excitation, 488 nm; emission, 500–550 nm), RFP (excitation, 554 nm; emission, 580–630 nm), and chlorophyll autofluorescence (excitation, 488 nm; emission, 630–670 nm). The laser intensity 5% and gain 10% were used to observe the chlorophyll autofluorescence, except that the SsCm1-GFP strains were inoculated onto N. benthamiana. For evaluating fluorescence intensity, gain and laser intensity were set uniformly and quantified using ImageJ.

BiFC assays were performed in N. benthamiana leaves. Agrobacterium mixtures carrying the appropriate BiFC constructs were infiltrated into N. benthamiana leaves. Samples were imaged with a Leica confocal microscope (Leica Microsystems) using LAS-X software with the preset settings for YFP (excitation, 514 nm; emission, 525–575 nm) at 2 dpi.

Co-IP and split-luciferase assays

Co-IP assays were performed as previously described⁵⁰. Briefly, 1 g of N. benthamiana leaf tissue was collected 2 d post infiltration with Agrobacterium and frozen in liquid nitrogen. Total proteins were extracted with protein extraction buffer (100 mM Tris-HCl pH 8, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 10 mM DTT, 2 mM PMSF, 10 mM NaF, 10 mM Na₂MoO₄, 2 mM NaVO₃, 1%(v/v) NP-40, 1%(v/v) plant protease inhibitor cocktail). Immunoprecipitation was performed with 40 µL of anti-GFP affinity beads 4FF (SA070005, Smart-Lifesciences) incubated for 1 h at 4 °C. The beads were washed 4 times with 1 mL cold wash buffer (100 mM Tris-HCl pH 8, 150 mM NaCl, 10% glycerol, 2 mM DTT, 10 mM NaF, 10 mM Na₂MoO₄, 2 mM NaVO₃, 0.5%(v/v) NP-40, 1%(v/v) plant protease inhibitor cocktail). Finally, the washed beads were resuspended in 100 µL SDS loading buffer and incubated for 10 min at 70 °C. The immunoprecipitated proteins and input proteins were separated on SDS-PAGE gels for western blotting with rabbit polyclonal anti-GFP (D110008, 1:5000; Sangon), rabbit polyclonal anti-FLAG (D110005, 1:2000; Sangon), and goat anti-rabbit IgG-HRP (A9169, 1:5000; Sigma) antibody. Chemiluminescence was imaged by Chemiluminescence Image System (Tanon) and blots were stained with CBB to verify equal loading.

Split-luciferase assays were performed as previously described⁵⁰. Briefly, Agrobacterium strains containing the desired plasmids were infiltrated into N. benthamiana leaves. CCD imaging was performed at 2 dpi. Leaves were infiltrated with 0.5 mM luciferin (HY-12591B, MCE) and kept in the dark for 5 min before CCD imaging. The images were taken with Chemiluminescence Image System (Tanon).

Protein stability assays

For the protein stability assays in the nonimmunity state, MORF2s-GFP or GmPETE-GFP co-expressed with SsCm1-FLAG or SsVSP25-FLAG in N. benthamiana were used. Total proteins were extracted 48 h after Agrobacterium-infiltration. For the protein stability assays during immune activation, N. benthamiana leaves were expressed with MORF2s-GFP alone (Control) or co-expressed with SsCm1-FLAG, and the leaves were treated 36 h after agroinfiltration. The treatments included infiltration with H₂O, 1 µM flg22, 200 µg/mL chitin, and 50 µg/mL SCFE, or inoculation with DC3000 (OD₆₀₀ = 0.0005), a 2*10⁶/mL spore suspension of B. cinerea, and mycelia of the ΔSsCm1-17 mutant. Total proteins were extracted 30 min after treatments were applied.

For cell free degradation assay, His-SsCm1 or His-SsVSP28 expressed in E. coli BL21 were purified and co-incubated with the total protein extracted from N. benthamiana leaves. After incubation for the indicated times, western blotting was performed.

For evaluate the stability of endogenous MORF2 proteins in plants. Firstly, the anti-MORF2 polyclonal antibodies were custom-developed by QiWei YiCheng Technology. Briefly, the synthesized AtMORF2_159-174aa peptide (LPDSYVDPENKDYGAE-C) was conjugated to keyhole limpet hemocyanin carrier to immunize rabbits. The rabbit polyclonal antiserum was purified by affinity chromatography using AtMORF2_159-174aa peptide, and the eluate was used as anti-MORF2 antibody. The applicability of antibody was evaluated by detecting MORF2 protein of Col-0, the morf2 lines, and 35S:GmMORF2a-GFP lines (Supplementary Fig. 14). The A. thaliana or soybean were treated according to the indicated treatment measures and times, and then the total protein was extracted.

For evaluate the stability of MORF2 protein in chloroplasts. Density gradient centrifugation was used to extract chloroplasts of Arabidopsis or soybean according to the manufacturer’s instructions of the Chloroplast Extraction Kit (PTE010, Coolaber). Subsequently, chloroplast proteins were extracted and incubated with about 50 µg of purified protein or SCFE for 2 h at 25 °C, then western blotting was performed. Chloroplast protein loading was indicated by detecting the protein content of rubisco large subunit (52.7 kDa, RbcL) without actin.

All protein samples were separated on SDS-PAGE gels for western blotting with mouse monoclonal anti-GFP (D191040, 1:5000; Sangon), rabbit polyclonal anti-FLAG (D110005, 1:2000; Sangon), mouse monoclonal anti-His (CW0286M, 1:1000; CWBIO), rabbit polyclonal anti-MORF2 (1:1000, this work), rabbit anti-RbcL (AS03037, 1:10000; Agrisera), rabbit polyclonal anti-actin (AS132640, 1:5000; Agrisera), goat anti-mouse IgG-HRP (D110087, 1:5000; Sangon), or goat anti-rabbit IgG-HRP (A9169, 1:5000; Sigma) antibodies. Chemiluminescence was imaged by Chemiluminescence Image System (Tanon) and blots were stained with CBB to verify equal loading. Protein abundance was quantified using ImageJ.

ROS staining, fluorescence quantification of CellROX, and ion leakage assays

For ROS staining of N. benthamiana, leaf discs from the area infiltrated with the designated Agrobacterium strains were collected 36 h after agroinfiltration. These leaf discs were vacuum-infiltrated with 1 mg/mL DAB (pH 5.5; D8001, Sigma) or 1 mg/mL NBT (A610379, Sangon) and incubated for 3 h at 28 °C. Chlorophyll was removed by boiling in ethanol (95% v/v) for 10 min. H₂O₂ production was visualized as a reddish-brown coloration based on DAB staining and superoxide was visualized as a dark blue-colored formazan deposit based on NBT staining. ROS staining in Arabidopsis was performed as previously described¹⁵. Briefly, 15 μM dexamethasone (D4902, Sigma) or 0.5 mM SA (S7401, Sigma) was infiltrated into leaves and kept in continuous light (100 μmol photons m⁻² s⁻¹) for 18 h or 24 h, respectively, and cellular H₂O₂ and superoxide were visualized by DAB and NBT staining, respectively.

Ion leakage assays were performed as previously described¹². Briefly, 15 μM dexamethasone, Pst-AvrRpt2 (OD₆₀₀ = 0.02), or 0.5 mM SA was infiltrated into leaves, which were kept in light for 36 h, 18 h or 36 h, respectively. Leaf discs were punched out and transferred to a tube containing 10 mL water. After 6 h of incubation at room temperature, conductivity of the solution (C₁) was measured with conductivity meter, then the conductivity (C₂) was measured after boiling for 10 min to completely kill the cells. The ion leakage ratio = (C₂ − C₁)/C₂*100%. For measuring the ion leakage of leaves after inoculation of S. sclerotiorum, pure hyphae were inoculated onto leaves, and then the area of inoculation site after designated inoculation time was collected for measurement.

For CellROX fluorescence quantification, the leaves were vacuum-infiltrated with 10 μM CellROX™ Deep Red (C10422, Invitrogen) for 30 min after different treatments at specific time points designed for the experiment. Leaf discs were placed in 96-well plates and fluorescence intensity was measured with a microplate reader (Excitation/Emission = 640/665 nm). For measuring the CellROX fluorescence of leaves after inoculation of S. sclerotiorum, pure hyphae were inoculated onto leaves, and then the area of inoculation site after designated inoculation time was collected for measurement.

Determination of Fv/Fm

The maximum photochemical efficiency of PSII (Fv/Fm) was determined with a FluorCam system (FC800-C/1010GFP, Photon Systems Instruments) containing a CCD camera and an irradiation system according to the instrument manufacturer’s instructions after different treatments for the specifically designated time points.

Quantification and statistical analysis

Statistical analyses were performed with the Prism 8.0 software (GraphPad). Details about the statistical methods and number of samples (n) were described in the relevant figure legends.

Accession numbers

Accession numbers and sequence information of the genes investigated in this study can be found in the S. sclerotiorum genome database (https://www.ncbi.nlm.nih.gov/assembly/GCF_000146945.2) under the following accession numbers: SsVSP30 (Sscle12g088370), SsCm1 (SsVSP31, Sscle12g088370), SsVSP32 (Sscle06g055310), Sstublin (Sscle02g015170), SsVSP25 (Sscle04g038020), SsNE2 (Sscle03g024000), SsCm2 (Sscle10g078330), and SsVSP28 (Sscle01g006330); in the N. benthamiana genome database (https://solgenomics.net/organism/Nicotiana_benthamiana/genome) under the following accession numbers: NbMORF2a (Niben261Chr13g0626014.1), NbMORF2b (Niben101Scf07015g00008.1), NbMORF2c (Niben101Scf07015g00010.1), and NbMORF9 (Niben101Scf00176g00005.1); in the G. max genome database (https://phytozome-next.jgi.doe.gov/info/Gmax_Wm82_a4_v1) under the following accession numbers: GmMORF2a (Glyma.20G206400), GmMORF2b (Glyma.10G183800), GmActin (Glyma.18G290800), GmPETE (Glyma.06G020400), GmCm1 (Glyma.13G059400), GmCm2 (Glyma.17G222700), and GmCm3 (Glyma.01G054500); and in the Arabidopsis TAIR database (https://www.arabidopsis.org) under the following accession numbers: ACT2 (AT3G18780), AtMORF2 (AT2G33430), FRK1 (AT2G19190), PR1 (AT2G14610), PR4 (AT3G04720), PAD3 (AT3G26830), ICS1 (AT1G74710), SARD1 (AT1G73805), MKK5 (AT3G21220), LHCB1.2 (AT1G29910), PsbA (ATCG00020), PETE (AT1G20340), PR2 (AT3G57260), and PAD4 (AT3G52430).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The authors declare that the data supporting the findings of this study are available within the article, Supplementary Information, Supplementary Data, and Source Data. Uncropped gels and blots are provided in Supplementary Fig. 18. Source data are provided with this paper.

References

Jones, J. D., Vance, R. E. & Dangl, J. L. Intracellular innate immune surveillance devices in plants and animals. Science 354, https://doi.org/10.1126/science.aaf6395 (2016).
Ngou, B. P. M., Ahn, H. K., Ding, P. & Jones, J. D. G. Mutual potentiation of plant immunity by cell-surface and intracellular receptors. Nature 592, 110–115 (2021).
Article ADS CAS Google Scholar
Peng, Y., van Wersch, R. & Zhang, Y. Convergent and divergent signaling in PAMP-triggered immunity and effector-triggered immunity. Mol. Plant Microbe Interact. 31, 403–409 (2018).
Article CAS Google Scholar
Yang, F., Xiao, K., Pan, H. & Liu, J. Chloroplast: the emerging battlefield in plant-microbe interactions. Front. Plant Sci. 12, 637853 (2021).
Article Google Scholar
Medina-Puche, L. et al. A defense pathway linking plasma membrane and chloroplasts and co-opted by pathogens. Cell 182, 1109–1124.e1125 (2020).
Article CAS Google Scholar
de Torres Zabala, M. et al. Chloroplasts play a central role in plant defence and are targeted by pathogen effectors. Nat. Plants 1, 15074 (2015).
Article Google Scholar
Nomura, H. et al. Chloroplast-mediated activation of plant immune signalling in Arabidopsis. Nat. Commun. 3, 926 (2012).
Article ADS Google Scholar
Zhu, J. K. Abiotic stress signaling and responses in plants. Cell 167, 313–324 (2016).
Article CAS Google Scholar
de Souza, A., Wang, J. Z. & Dehesh, K. Retrograde signals: integrators of interorganellar communication and orchestrators of plant development. Annu. Rev. Plant Biol. 68, 85–108 (2017).
Article Google Scholar
Chan, K. X., Phua, S. Y., Crisp, P., McQuinn, R. & Pogson, B. J. Learning the languages of the chloroplast: retrograde signaling and beyond. Annu. Rev. Plant Biol. 67, 25–53 (2016).
Article CAS Google Scholar
Guo, H. et al. Plastid-nucleus communication involves calcium-modulated MAPK signalling. Nat. Commun. 7, 12173 (2016).
Article ADS Google Scholar
Lee, K. P. et al. Hierarchical regulatory module GENOMES UNCOUPLED1-GOLDEN2-LIKE1/2-WRKY18/40 modulates salicylic acid signaling. Plant Physiol. 192, 3120–3133 (2023).
Article ADS CAS Google Scholar
Laloi, C. et al. Cross-talk between singlet oxygen- and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 104, 672–677 (2007).
Article ADS CAS Google Scholar
Serrano, I., Audran, C. & Rivas, S. Chloroplasts at work during plant innate immunity. J. Exp. Bot. 67, 3845–3854 (2016).
Article CAS Google Scholar
Su, J. et al. Active photosynthetic inhibition mediated by MPK3/MPK6 is critical to effector-triggered immunity. PLoS Biol. 16, e2004122 (2018).
Article Google Scholar
Grant, M. R. & Jones, J. D. Hormone (dis)harmony moulds plant health and disease. Science 324, 750–752 (2009).
Article ADS CAS Google Scholar
Mühlenbock, P. et al. Chloroplast signaling and LESION SIMULATING DISEASE1 regulate crosstalk between light acclimation and immunity in Arabidopsis. Plant Cell 20, 2339–2356 (2008).
Article Google Scholar
Zurbriggen, M. D. et al. Chloroplast-generated reactive oxygen species play a major role in localized cell death during the non-host interaction between tobacco and Xanthomonas campestris pv. vesicatoria. Plant J. 60, 962–973 (2009).
Article CAS Google Scholar
Trotta, A., Rahikainen, M., Konert, G., Finazzi, G. & Kangasjärvi, S. Signalling crosstalk in light stress and immune reactions in plants. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 369, 20130235 (2014).
Google Scholar
Ballaré, C. L. Light regulation of plant defense. Annu. Rev. Plant Biol. 65, 335–363 (2014).
Article Google Scholar
Göhre, V., Jones, A. M., Sklenář, J., Robatzek, S. & Weber, A. P. Molecular crosstalk between PAMP-triggered immunity and photosynthesis. Mol. Plant Microbe Interact. 25, 1083–1092 (2012).
Article Google Scholar
Bilgin, D. D. et al. Biotic stress globally downregulates photosynthesis genes. Plant Cell Environ. 33, 1597–1613 (2010).
Article CAS Google Scholar
Lv, R. et al. Uncoupled expression of nuclear and plastid photosynthesis-associated genes contributes to cell death in a lesion mimic mutant. Plant Cell 31, 210–230 (2019).
Article CAS Google Scholar
Duan, J. et al. Impaired PSII proteostasis promotes retrograde signaling via salicylic acid. Plant Physiol. 180, 2182–2197 (2019).
Article CAS Google Scholar
Xu, Q. et al. An effector protein of the wheat stripe rust fungus targets chloroplasts and suppresses chloroplast function. Nat. Commun. 10, 5571 (2019).
Article ADS CAS Google Scholar
Tang, L. et al. An effector of a necrotrophic fungal pathogen targets the calcium-sensing receptor in chloroplasts to inhibit host resistance. Mol. Plant Pathol. 21, 686–701 (2020).
Article CAS Google Scholar
Zhou, J., Zeng, L., Liu, J. & Xing, D. Manipulation of the xanthophyll cycle increases plant susceptibility to Sclerotinia sclerotiorum. PLoS Pathog. 11, e1004878 (2015).
Article Google Scholar
Yang, C., Zhang, Z., Gao, H., Liu, M. & Fan, X. Mechanisms by which the infection of Sclerotinia sclerotiorum (Lib.) de Bary affects the photosynthetic performance in tobacco leaves. BMC Plant Biol. 14, 240 (2014).
Article Google Scholar
Bolton, M. D., Thomma, B. P. & Nelson, B. D. Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. Mol. Plant Pathol. 7, 1–16 (2006).
Article CAS Google Scholar
Liang, X. & Rollins, J. A. Mechanisms of broad host range necrotrophic pathogenesis in Sclerotinia sclerotiorum. Phytopathology 108, 1128–1140 (2018).
Article CAS Google Scholar
Williams, B., Kabbage, M., Kim, H. J., Britt, R. & Dickman, M. B. Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment. PLoS Pathog. 7, e1002107 (2011).
Article CAS Google Scholar
Cessna, S. G., Sears, V. E., Dickman, M. B. & Low, P. S. Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant. Plant Cell 12, 2191–2200 (2000).
Article CAS Google Scholar
Kabbage, M., Williams, B. & Dickman, M. B. Cell death control: the interplay of apoptosis and autophagy in the pathogenicity of Sclerotinia sclerotiorum. PLoS Pathog. 9, e1003287 (2013).
Article CAS Google Scholar
Xu, L., Li, G., Jiang, D. & Chen, W. Sclerotinia sclerotiorum: an evaluation of virulence theories. Annu. Rev. Phytopathol. 56, 311–338 (2018).
Article CAS Google Scholar
Derbyshire, M. et al. The complete genome sequence of the phytopathogenic fungus Sclerotinia sclerotiorum reveals insights into the genome architecture of broad host range pathogens. Genome Biol. Evol. 9, 593–618 (2017).
Article CAS Google Scholar
Lyu, X. et al. A small secreted virulence-related protein is essential for the necrotrophic interactions of Sclerotinia sclerotiorum with its host plants. PLoS Pathog. 12, e1005435 (2016).
Article Google Scholar
Yang, G. et al. A cerato-platanin protein SsCP1 targets plant PR1 and contributes to virulence of Sclerotinia sclerotiorum. N. Phytol. 217, 739–755 (2018).
Article CAS Google Scholar
Wei, W. et al. A fungal extracellular effector inactivates plant polygalacturonase-inhibiting protein. Nat. Commun. 13, 2213 (2022).
Article ADS CAS Google Scholar
Djamei, A. et al. Metabolic priming by a secreted fungal effector. Nature 478, 395–398 (2011).
Article ADS CAS Google Scholar
Westrick, N. M. et al. Gene regulation of Sclerotinia sclerotiorum during infection of Glycine max: on the road to pathogenesis. BMC Genomics 20, 157 (2019).
Article Google Scholar
Guyon, K., Balagué, C., Roby, D. & Raffaele, S. Secretome analysis reveals effector candidates associated with broad host range necrotrophy in the fungal plant pathogen Sclerotinia sclerotiorum. BMC Genomics 15, 336 (2014).
Article Google Scholar
Li, J. et al. Introduction of large sequence inserts by CRISPR-Cas9 to create pathogenicity mutants in the multinucleate filamentous pathogen Sclerotinia sclerotiorum. mBio 9, https://doi.org/10.1128/mBio.00567-18 (2018).
Schnappauf, G., Lipscomb, W. N. & Braus, G. H. Separation of inhibition and activation of the allosteric yeast chorismate mutase. Proc. Natl. Acad. Sci. USA 95, 2868–2873 (1998).
Article ADS CAS Google Scholar
Takenaka, M. et al. Multiple organellar RNA editing factor (MORF) family proteins are required for RNA editing in mitochondria and plastids of plants. Proc. Natl. Acad. Sci. USA 109, 5104–5109 (2012).
Article ADS CAS Google Scholar
Lorrain, S., Vailleau, F., Balagué, C. & Roby, D. Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?. Trends Plant Sci. 8, 263–271 (2003).
Article CAS Google Scholar
Yuan, J. et al. Two chloroplast-localized MORF proteins act as chaperones to maintain tetrapyrrole biosynthesis. N. Phytol. 235, 1868–1883 (2022).
Article CAS Google Scholar
Ren, D., Yang, H. & Zhang, S. Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis. J. Biol. Chem. 277, 559–565 (2002).
Article CAS Google Scholar
Seifbarghi, S. et al. Receptor-like kinases BAK1 and SOBIR1 are required for necrotizing activity of a novel group of Sclerotinia sclerotiorum necrosis-inducing effectors. Front. Plant Sci. 11, 1021 (2020).
Article Google Scholar
Kamoun, S., van West, P., Vleeshouwers, V. G., de Groot, K. E. & Govers, F. Resistance of Nicotiana benthamiana to Phytophthora infestans is mediated by the recognition of the elicitor protein INF1. Plant Cell 10, 1413–1426 (1998).
Article CAS Google Scholar
Yu, G. et al. A bacterial effector protein prevents MAPK-mediated phosphorylation of SGT1 to suppress plant immunity. PLoS Pathog. 16, e1008933 (2020).
Article CAS Google Scholar
Todesco, M. et al. Natural allelic variation underlying a major fitness trade-off in Arabidopsis thaliana. Nature 465, 632–636 (2010).
Article ADS CAS Google Scholar
Huot, B., Yao, J., Montgomery, B. L. & He, S. Y. Growth-defense tradeoffs in plants: a balancing act to optimize fitness. Mol. Plant 7, 1267–1287 (2014).
Article CAS Google Scholar
Zhang, Y., Tian, L. & Lu, C. Chloroplast gene expression: Recent advances and perspectives. Plant Commun. 4, 100611 (2023).
Article CAS Google Scholar
García-Andrade, J., Ramírez, V., López, A. & Vera, P. Mediated plastid RNA editing in plant immunity. PLoS Pathog. 9, e1003713 (2013).
Article Google Scholar
Ling, Q. & Jarvis, P. Regulation of chloroplast protein import by the ubiquitin E3 ligase SP1 is important for stress tolerance in plants. Curr. Biol. 25, 2527–2534 (2015).
Article CAS Google Scholar
Wang, S. et al. YR36/WKS1-mediated phosphorylation of PsbO, an extrinsic member of photosystem II, inhibits photosynthesis and confers stripe rust resistance in Wheat. Mol. Plant 12, 1639–1650 (2019).
Article CAS Google Scholar
Liu, M. et al. Phosphorylation-guarded light-harvesting complex II contributes to broad-spectrum blast resistance in rice. Proc. Natl. Acad. Sci. USA 116, 17572–17577 (2019).
Article ADS CAS Google Scholar
Wang, L. et al. Singlet oxygen- and EXECUTER1-mediated signaling is initiated in grana margins and depends on the protease FtsH2. Proc. Natl. Acad. Sci. USA 113, E3792–E3800 (2016).
CAS Google Scholar
Kim, C. et al. Chloroplasts of Arabidopsis are the source and a primary target of a plant-specific programmed cell death signaling pathway. Plant Cell 24, 3026–3039 (2012).
Article CAS Google Scholar
Li, M. & Kim, C. Chloroplast ROS and stress signaling. Plant Commun. 3, 100264 (2022).
Article CAS Google Scholar
Triantaphylidès, C. & Havaux, M. Singlet oxygen in plants: production, detoxification and signaling. Trends Plant Sci. 14, 219–228 (2009).
Article Google Scholar
Yang, Y. et al. Cytidine-to-Uridine RNA editing factor NbMORF8 negatively regulates plant immunity to Phytophthora pathogens. Plant Physiol. 184, 2182–2198 (2020).
Article CAS Google Scholar
Yapa, M. M., Doroodian, P., Gao, Z., Yu, P. & Hua, Z. MORF2-mediated plastidial retrograde signaling is involved in stress response and skotomorphogenesis beyond RNA editing. Front. Plant Sci. 14, 1146922 (2023).
Article Google Scholar
Zhang, F. et al. Tetrapyrrole biosynthetic enzyme protoporphyrinogen IX oxidase 1 is required for plastid RNA editing. Proc. Natl. Acad. Sci. USA 111, 2023–2028 (2014).
Article ADS CAS Google Scholar
Geng, R. et al. PROGRAMMED CELL DEATH8 interacts with tetrapyrrole biosynthesis enzymes and ClpC1 to maintain homeostasis of tetrapyrrole metabolites in Arabidopsis. N. Phytol. 238, 2545–2560 (2023).
Article CAS Google Scholar
Zhao, X., Huang, J. & Chory, J. GUN1 interacts with MORF2 to regulate plastid RNA editing during retrograde signaling. Proc. Natl. Acad. Sci. USA 116, 10162–10167 (2019).
Article ADS CAS Google Scholar
Coego, A. et al. An Arabidopsis homeodomain transcription factor, OVEREXPRESSOR OF CATIONIC PEROXIDASE 3, mediates resistance to infection by necrotrophic pathogens. Plant Cell 17, 2123–2137 (2005).
Article CAS Google Scholar
Gao, L. L., Hong, Z. H., Wang, Y. & Wu, G. Z. Chloroplast proteostasis: a story of birth, life, and death. Plant Commun. 4, 100424 (2023).
Article CAS Google Scholar
Woodson, J. D. Chloroplast stress signals: regulation of cellular degradation and chloroplast turnover. Curr. Opin. Plant Biol. 52, 30–37 (2019).
Article CAS Google Scholar
Wang, S. & Blumwald, E. Stress-induced chloroplast degradation in Arabidopsis is regulated via a process independent of autophagy and senescence-associated vacuoles. Plant Cell 26, 4875–4888 (2014).
Article CAS Google Scholar
Mohd Ali, S. et al. Multiple ubiquitin E3 ligase genes antagonistically regulate chloroplast-associated protein degradation. Curr. Biol. 33, 1138–1146.e1135 (2023).
Article CAS Google Scholar
Ling, Q., Huang, W., Baldwin, A. & Jarvis, P. Chloroplast biogenesis is regulated by direct action of the ubiquitin-proteasome system. Science 338, 655–659 (2012).
Article ADS CAS Google Scholar
Woodson, J. D. et al. Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts. Science 350, 450–454 (2015).
Article ADS CAS Google Scholar
Wu, J. et al. Solid-like condensation of MORF8 inhibits RNA editing under heat stress in Arabidopsis. Nat. Commun. 16, 2789 (2025).
Article CAS Google Scholar
Bauters, L. et al. Chorismate mutase and isochorismatase, two potential effectors of the migratory nematode Hirschmanniella oryzae, increase host susceptibility by manipulating secondary metabolite content of rice. Mol. Plant Pathol. 21, 1634–1646 (2020).
Article CAS Google Scholar
He, Q. et al. A novel chorismate mutase from Erysiphe quercicola performs dual functions of synthesizing amino acids and inhibiting plant salicylic acid synthesis. Microbiol. Res. 242, 126599 (2021).
Article CAS Google Scholar
Wen, Z. et al. Integrating GWAS and gene expression data for functional characterization of resistance to white mould in soyabean. Plant Biotechnol. J. 16, 1825–1835 (2018).
Article CAS Google Scholar
Mengiste, T. Plant immunity to necrotrophs. Annu. Rev. Phytopathol. 50, 267–294 (2012).
Article CAS Google Scholar
Oh, S. K. et al. In planta expression screens of Phytophthora infestans RXLR effectors reveal diverse phenotypes, including activation of the Solanum bulbocastanum disease resistance protein Rpi-blb2. Plant Cell 21, 2928–2947 (2009).
Article CAS Google Scholar
Zhang, W. et al. Arabidopsis receptor-like protein 30 and receptor-like kinase suppressor of BIR1- 1/EVERSHED mediate innate immunity to necrotrophic fungi. Plant Cell 25, 4227–4241 (2013).
Article CAS Google Scholar
Xiao, K. et al. The Snf5-Hsf1 transcription module synergistically regulates stress responses and pathogenicity by maintaining ROS homeostasis in Sclerotinia sclerotiorum. N. Phytol. 241, 1794–1812 (2024).
Article CAS Google Scholar
Sooksai, T. et al. Production of cutinase from Fusarium falciforme and its application for hydrophilicity improvement of polyethylene terephthalate fabric. 3 Biotech 9, 389 (2019).
Article Google Scholar
Lyu, X. et al. GmCRY1s modulate gibberellin metabolism to regulate soybean shade avoidance in response to reduced blue light. Mol. Plant 14, 298–314 (2021).
Article CAS Google Scholar
Gilchrist, D. G. & Connelly, J. A. Chorismate mutase from mung bean and sorghum. Methods Enzymol. 142, 450–463 (1987).
Article CAS Google Scholar

Download references

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (32172505 to J.L., 32272484 to H.P., 323B2055 to K.X.), the National Key Research and Development Program of China (2019YFE0114200 to H.P.), and the Natural Science Foundation of Jilin Province (20230101156JC to J.L.). We thank Professor Fengjie Sun of Georgia Gwinnett College, for help with editing the paper.

Author information

These authors contributed equally: Kunqin Xiao, Feng Yang.

Authors and Affiliations

College of Plant Sciences, Jilin University, Changchun, China
Kunqin Xiao, Feng Yang, Wenjing Cui, Anmo Li, Jinxin Guo, Fengting Wang, Xun Xu, Yanhua Zhang, Xianghui Zhang, Jinliang Liu & Hongyu Pan
State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, China
Feng Yang & Xiaojie Wang
Department of Plant Pathology, University of Florida, Gainesville, FL, USA
Jeffrey A. Rollins
Chongqing Key Laboratory of Plant Disease Biology, College of Plant Protection, Southwest University, Chongqing, China
Xinhua Sun

Authors

Kunqin Xiao
View author publications
Search author on:PubMed Google Scholar
Feng Yang
View author publications
Search author on:PubMed Google Scholar
Wenjing Cui
View author publications
Search author on:PubMed Google Scholar
Anmo Li
View author publications
Search author on:PubMed Google Scholar
Jeffrey A. Rollins
View author publications
Search author on:PubMed Google Scholar
Jinxin Guo
View author publications
Search author on:PubMed Google Scholar
Xinhua Sun
View author publications
Search author on:PubMed Google Scholar
Fengting Wang
View author publications
Search author on:PubMed Google Scholar
Xiaojie Wang
View author publications
Search author on:PubMed Google Scholar
Xun Xu
View author publications
Search author on:PubMed Google Scholar
Yanhua Zhang
View author publications
Search author on:PubMed Google Scholar
Xianghui Zhang
View author publications
Search author on:PubMed Google Scholar
Jinliang Liu
View author publications
Search author on:PubMed Google Scholar
Hongyu Pan
View author publications
Search author on:PubMed Google Scholar

Contributions

H.P. and J.L. directed and supervised the research. H.P., J.L., and K.X. designed the research. K.X. and F.Y. conducted most of the experiments, analyzed the data, and wrote the original draft. W.C., J.G., and A.L. assisted with the qRT-PCR experiments and the acquisition of transgenic materials, A.L. and X.X. generated the CRISPR/Cas9-engineered Gmmorf2a/b mutants, and X.S. performed the luminol-based ROS burst assays and the subcellular co-location assays. F.W., Y.Z., and X.Z. assisted in writing. J.A.R., J.L., X.W., and H.P. revised the manuscript.

Corresponding authors

Correspondence to Jinliang Liu or Hongyu Pan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

Supplementary Data 1

Reporting Summary

Transparent Peer Review file

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Cite this article

Xiao, K., Yang, F., Cui, W. et al. A fungal effector promotes infection via stabilizing a negative regulatory factor of chloroplast immunity. Nat Commun 16, 6970 (2025). https://doi.org/10.1038/s41467-025-62326-4

Download citation

Received: 01 March 2024
Accepted: 14 July 2025
Published: 29 July 2025
DOI: https://doi.org/10.1038/s41467-025-62326-4