Fig. 4: RARE destabilizes RRS1/RPS4 complex in a RRS1-dependent manner and negatively regulates RRS1/RPS4–mediated defense responses. | Nature Communications

Fig. 4: RARE destabilizes RRS1/RPS4 complex in a RRS1-dependent manner and negatively regulates RRS1/RPS4–mediated defense responses.

From: Reversible ubiquitination conferred by domain shuffling controls paired NLR immune receptor complex homeostasis in plant immunity

Fig. 4

A RARE directly destabilizes RRS1 and indirectly destabilizes RPS4 in the presence of RRS1. RRS1-R, RPS4 or RRS1-R/RPS4 were transiently co-expressed with RARE in Nb at 2 dpi and then subjected to immunoblotting with anti-FLAG antibody for detecting RRS1-R/RPS4 or anti-HA antibody for detecting RARE. The HA-tagged GFP served as a control. B RARE modulates RPS4 homeostasis via RRS1. RPS4-FLAG was co-expressed together with RARE-HA or GFP-HA in protoplasts from Col-0 or rrs-1 plants. RPS4-FLAG proteins were detected by immunoblotting with anti-FLAG antibody. C Immunoblot analyses of RRS1-R and RPS4 protein levels in Col-0, rare, and RAREOE background. Total proteins were extracted from 10-days-old seedlings and subjected to immunoblotting using anti-FLAG antibody for detecting RRS1-R and anti-HA antibody for detecting RPS4, respectively. Ponceau S staining of Rubisco indicates equal loading. D Suppression of RRS1/RPS4-mediated cell death upon AvrRps4 perception in tobacco plants. Each leaf section was transiently coinfiltrated with RRS1-R/RPS4, AvrRps4, and either GFP or RARE. Photographs assessing HR were taken 4 dpi. E Overexpression of RARE suppresses the autoimmune phenotype triggered by RRS1-Rslh1 and RRS1-RT1214A, the auto-active alleles of RRS1-R. Morphology of 5-week-old soil-grown plants of Col-0, RRS1-Rslh1, RRS1-RT1214A, RAREOERRS1-Rslh1 and RAREOERRS1-RT1214A. Scale bar, 0.5 cm. F Relative expression levels of PR1 and PR2 were determined by real-time PCR. The relative transcript levels of PR1 and PR2 were normalized to the expression of UBQ. Data are shown as means ± SD (n = 3 biological replicates). G RAREOE lines are compromised in RRS1/RPS4-mediated resistance against Pst DC3000(AvrRps4). 5-week-old plants were infiltrated with the bacteria at OD600 = 0.0005. Leaf disks within the infiltrated area were taken at days 0 and 3 to measure the bacterial growth. H Introduction of the RRS1-R transgene into the RAREOE#3 line restores disease resistance to Pst DC3000(AvrRps4). Note that the epitope-tagged RRS1-R/RPS4 transgenic line#4 was used to cross with RAREOE transgenic line. For (G–H), data are shown as means ± SD (n = 4 and 6 biological replicates, respectively). For (F–H), the corresponding p values can be found in the Source Data.

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