Background & Summary

Since its description as a pest of tobacco in Greece1, the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) has been a persistent scourge to field and greenhouse-grown agricultural crops, fibre commodities, and ornamental plants around the world2. In the United States, the 1980’s invasion of B. tabaci B biotype3, now often referred to as the Middle East–Asia Minor 1 (MEAM1)4 (also mitotype B or Bemisia argentifolii Bellows & Perring5,6), resulted in hundreds of millions of dollars in economic losses. Plant damage associated with rapid increases in the whitefly population, extreme polyphagy7, physiological disorders due to direct feeding8,9, and, most seriously, virus transmission10, were attributed to the new invader. Approximately 40 years later, B. tabaci continues to pose substantial problems for agriculturalists and subsistence farmers throughout the world, mainly due to the transmission of viruses, including begomoviruses (family Geminiviridae) which severely limit yield in numerous vegetable11, root12, and fibre crops13.

Begomoviruses are dicotyledonous plant viruses with single-stranded, mono- or bipartite circular genomes approximately 2.7 kilobases in length14. Viruses in this genus are spread exclusively by members of the B. tabaci cryptic species complex in a persistent, circulative manner15. Differential transmission of certain begomoviruses has been reported for different species within the complex16,17,18. The route viral particles take within the whitefly vector is complex19. The whitefly uses its stylets to ingest virions from phloem sap of an infected host, which then pass through the food canal, cibarium, and esophagus before entering the midgut filter chamber. The filter chamber is the main site where virions concentrate and are translocated into the haemolymph20,21,22. Once in the haemolymph, viral particles are protected en route to the primary salivary glands by complexing with a GroEL chaperone protein manufactured by the whitefly endosymbiotic bacterium Hamiltonella in MEAM123,24 and Arsenophonus in Asia II-1 (also referred to as biotype/mitotypes K, P, ZHJ2, PCG-1, SY, PK14,25). Virions are then internalized into the salivary glands and finally egested back into the plant phloem, where the cycle starts anew.

One of the most serious viral pathogens transmitted by B. tabaci is tomato yellow leaf curl virus (TYLCV)26, a monopartite begomovirus that causes tomato yellow leaf curl disease (TYLCD)27. The onset and severity of TYLCD symptoms are highly variable and dependent on the tomato cultivar, timing of inoculation relative to plant senescence, and environmental conditions. Typical TYLCD symptoms include upward curling and yellowing of leaflet margins, reduced size of leaflets, stunting, aborted blooms, reduced fruit size, and irregular fruit ripening8,28. Complete yield losses due to TYLCD are not uncommon27. Due to this notoriety, TYLCV is one of the most studied begomoviruses transmitted by B. tabaci, and developing effective management strategies to reduce TYLCV-induced economic losses is an active area of research29,30,31.

A promising research avenue to reduce the deleterious effects of TYLCV on tomato production is the adoption of genomics-based technologies. This has manifested as the identification of genetic loci that confer resistance to TYLCV and their integration into stable tomato breeding lines32,33,34, as well as the development of RNA interference (RNAi) technologies targeting genes in the vector that affect different life history parameters (e.g., longevity, fecundity, etc.) and virus transmission35,36,37,38. Target genes for RNAi may be identified through transcriptomic studies that evaluate the response of the whitefly to begomovirus acquisition. This is accomplished by measuring the number of differentially expressed genes (DEGs) between viruliferous and non-viruliferous whiteflies after they are allowed to feed on a host with or without the pathogen of interest. Coupled with a well-annotated genome assembly39,40,41, the DEGs can be identified, and their functions determined, to select the best candidates for downstream control applications (e.g., RNAi, CRISPR)42,43.

Several studies have examined differential gene expression in B. tabaci following begomovirus acquisition44,45,46,47,48,49,50,51,52,53,54,55,56,57, but relatively few have investigated the transcriptional response of B. tabaci MEAM1 to TYLCV acquisition. Those that have reported substantially different numbers of DEGs, which is most likely a result of key methodological differences including the use of propagative material (i.e., infected plant cuttings) instead of potted plants49, the sequencing of only one tissue type (e.g., gut tissue)48, and the implementation of a gut-clearing step following the TYLCV acquisition access period (AAP)54. In this study, we examined the transcriptional response of B. tabaci to TYLCV after three AAPs (12, 36, and 60 h) on potted tomato followed by a gut-clearing step on a TYLCV non-host, collard (Brassica oleracea). The gut-clearing step was implemented to minimize any potential indirect effects that feeding on infected plant tissue has on the vector44,54, thereby increasing the likelihood that changes in gene expression profiles between viruliferous and non-viruliferous whiteflies directly reflect virus-vector interactions. We also took a novel approach to preparing the sequenced RNA-seq libraries as compared to previous studies of the vector transcriptional response to begomovirus acquisition. To date, rRNA depleted RNA-seq libraries have not been reported in similar studies of whitefly-virus interactions. Previous studies have demonstrated that gene quantification variation is dependent on RNA sample preparation, with rRNA depletion able to capture more unique and rare transcripts than poly(A) selection58,59,60. Such transcripts include those encoding replication-dependent histone proteins61 as well as long-noncoding RNAs62. This dataset will be useful to researchers interested in studying novel B. tabaci genetic targets for developing enhanced genomics-informed whitefly and whitefly-transmitted virus management.

Methods

Whitefly colony

A colony of B. tabaci MEAM1 was established in 2022 from adult whiteflies collected on field grown zucchini (Cucurbita pepo L.) in the research fields of the Coastal Research and Education Center in Charleston, South Carolina, USA. Whiteflies were then reared on collard (Brassica oleracea var. acephala) in a greenhouse (26 ± 4 °C) at the USDA-ARS, U. S. Vegetable Laboratory, with fresh collard plants added to the colony as needed.

Plant inoculations

To produce viruliferous whiteflies for use in plant inoculations, approximately 20 mating pairs of the MEAM1 colony maintained on collard were placed in clip cages and transferred to visibly symptomatic tomato plants (Solanum lycopersicum L., cultivar ‘Moneymaker’) infected with TYLCV for a 72-hour acquisition access period (AAP). Healthy tomato plants with at least three fully expanded true leaves were then subjected to feeding by approximately 20 mating pairs of non-viruliferous (healthy) or TYLCV-viruliferous (virus) whiteflies in clip cages for a 72-hour inoculation access period (IAP) to generate mock-inoculated (whitefly-exposed) and TYLCV-infected plants for use in experiments 3–4 weeks later. Plant infection status was assessed with end-point PCR before the initiation of feeding assays using primer pair KL14-324 (5′-CTTCGACAGCCCATACAGCA-3′) and KL14-325 (5′-GAGGGCCCACCAATAACTGT-3′) designed by Dr. Daniel Hasegawa in the laboratory of Dr. Kai-Shu Ling (USDA, ARS, U.S. Vegetable Laboratory, Charleston, SC, USA).

Feeding assays

To acquire tomato-acclimated, age-specific (staged) whiteflies for use in experiments, older collard leaves from the whitefly colony bearing 4th instar whitefly nymphs, with adult whiteflies removed, were excised and added to cages containing virus-free tomato plants for a five-day emergence period. On day five, the emerged adult whiteflies were aspirated into standard insect collection vials and released onto the mock-inoculated or TYLCV-infected tomato plants. Approximately 1,500 adult whiteflies (≤ five days post-emergence) were added to each experimental plant, which were held in separate BugDorm cages (60 × 60 × 60 cm; MegaView Science Co., Ltd., Taichung, Taiwan) in an insectary maintained at 26 ± 4 °C, 45 ± 15% RH, and a natural light/dark cycle (14:10 L:D). Whiteflies were allowed to feed for three acquisition access periods (AAPs): 12, 36, and 60 hours. At the end of each AAP, approximately 500 adult whiteflies from each experimental cage were transferred to collard, a non-host of TYLCV, for a 12-hr gut clearing period. The gut clearing step was added to ensure that differential expression was due to virus ingestion and acquisition rather than a response to feeding on TYLCV-infected plant tissue. Following gut clearing, all whiteflies were collected and frozen at −80 °C until RNA extraction. The experiment was replicated three times, and each replication consisted of one plant per treatment.

RNA extraction, Illumina library preparation, and RNA sequencing

Total RNA was extracted from whiteflies using TRIzol® with the PureLink® RNA Mini Kit following the manufacturer’s recommended protocol, except that initial sample lysis was conducted in 100 µL TRIzol® using an ice-cold pestle and the optional on-column DNase treatment protocol was performed. Illumina library preparation and RNA sequencing were performed at the Genomics Core Facility of Michigan State University (East Lansing, MI, USA). Whitefly and bacterial (symbiont) ribosomal RNA (rRNA) was depleted from samples using the QIAseq FastSelect rRNA Fly and 5S/16S/23S kits, respectively (Qiagen, Hilden, Germany). Stranded RNA-seq libraries were prepared using the Illumina Stranded Total RNA Library Preparation Ligation Kit (Illumina, Inc., San Diego, CA, USA) with Integrated DNA Technologies Unique Dual Index adapters following the manufacturer’s recommendations, except that half volume reactions were used. Library quality control and quantification was conducted using a combination of Qubit dsDNA HS (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Agilent 4200 TapeStation HS DNA1000 assays (Agilent Technologies, Santa Clara, CA, USA). The libraries were pooled in equimolar proportions and quantified using an Invitrogen Collibri Quantification qPCR kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sequencing was performed on an Illumina NovaSeq6000 sequencing system. The pool was loaded onto one lane of an Illumina S4 flow cell, and sequencing was performed in a 2×150bp paired-end format using a NovaSeq v1.5, 300 cycle reagent kit. Base calling was done by Illumina Real Time Analysis (RTA) v3.4.4, and the output of RTA was demultiplexed and converted to FASTQ format with Illumina Bcl2fastq (v2.20.0).

RNA-seq read processing, mapping, and differential expression analyses

Raw reads from the 18 RNA-seq libraries (six treatments with three replicates) were processed with fastp63 (v.0.12.4) to remove sequencing adapters, trim low-quality bases, and remove reads less than 75 base pairs (bp) in length. The cleaned reads were then aligned to the MEAM1 mitochondrial genome (NCBI Reference Sequence: NC_006279.1), three bacterial endosymbiont genomes (Candidatus Portiera aleyrodidarum, Candidatus Hamiltontella defensa, and Rickettsia sp. available at http://www.whiteflygenomics.org/ftp/MEAM1/endosymbionts/), and Release 138.1 of the SILVA rRNA database (SSU and LSU NR 99 Ref sequences; accessed on 26 June 2023) using HISAT264 (v.2.2.1). The aligned reads were filtered out, and the remaining reads were aligned to the MEAM1 nuclear genome assembly39 with STAR65 (v.2.7.10b) using the splice site information included in the MEAM1 general feature format file at http://www.whiteflygenomics.org/ftp/MEAM1/v1.2/MEAM1_v1.2.gff3.gz. The resulting SAM files were converted to BAM format with samtools66 (v.1.17). Differential expression (DE) analyses on read- and gene-level count matrices were performed with DESeq. 267 (v.1.40.2) in R68. The count matrix of sequencing reads unambiguously mapped to a gene (hereafter referred to as read mapping; RM) was generated using the BAM files mentioned previously. The gene-level count matrix (hereafter referred to as transcript quantification; TQ) was created with Salmon69 (v.1.6.0) and imported to R using the tximport package70 (v.1.28.0). In both RM and TQ analyses, genes with an adjusted p-value < 0.1 and minimum fold change of 1.2 were considered as differentially expressed. The illustrations in Fig. 1 were created using the package ggplot271 (v.4.4.1) in R.

Fig. 1
figure 1

Bar chart of the number of differentially expressed genes (DEGs) at each acquisition access period (AAP) identified by read mapping (RM) and transcript quantification (TQ).

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On average, 32.5 million cleaned RNA-seq reads were generated per library (N = 18), and 96.1% of the cleaned reads in each library aligned to the MEAM1 nuclear genome assembly (Table 1). Approximately four million more RNA-seq reads were generated per library in viruliferous (36.6 million) vs non-viruliferous whiteflies (32.6 million).

Table 1 Summary of ribodepleted RNA-Seq datasets generated from Bemisia tabaci MEAM1 whiteflies fed for 12, 36, or 60 h on healthy (virus free) and tomato yellow leaf curl virus infected plants followed by 12 h of gut clearing on collard.
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Thirty-seven DEGs were identified across all time points and types of analysis (Fig. 1; Table 2). Thirteen and 26 DEGs were identified by RM and TQ, respectively. Two genes (Bta05741, Bta05749) were identified as differentially expressed in both analyses, and no genes were differentially expressed at more than one time point. In RM analyses, 10 genes were upregulated, and 3 genes were downregulated. Two of the genes upregulated at 72 hours (Bta13103, Bta13961) were horizontally acquired from plants72. In TQ analyses, 8 genes were upregulated, and 18 genes were downregulated.

Table 2 Differentially expressed genes in Bemisia tabaci MEAM1 fed for 12, 36, and 60 h on healthy (virus free) and tomato yellow leaf curl virus infected plants followed by 12 h of gut clearing on collard. GeneIDs followed by an asterisk were identified as differentially expressed in both analyses.
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Data Records

The data associated with this project have been accessioned with the National Center for Biotechnology Information (NCBI) under BioProject number PRJNA1096732: Transcriptomics of viruliferous and non-viruliferous Bemisia tabaci MEAM173. Illumina RNA-seq reads have been deposited in the NCBI Sequence Read Archive under accession numbers SRR28578498–SRR28578515, and the BioSamples used to generate the RNA-seq reads have been assigned BioSample accession numbers SAMN40761531– SAMN40761536.

Technical Validation

Total RNA quantification was performed using a DeNovix DS-11 FX spectrophotometer/fluorometer (DeNovix Inc., Wilmington, DE, USA) using 1 µL of total RNA. RNA integrity was assessed on a 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA) at the Molecular Analytics Core, part of the Department of Regenerative Medicine and Cell Biology at the Medical University of South Carolina (Charleston, SC, USA). Cohorts of six (non-viruliferous) and 10 (viruliferous) whiteflies from each replicate and time point were collected and individually extracted to screen for the absence or presence of TYLCV. All whiteflies in the non-viruliferous treatments tested negative for TYLCV at each time point. In the viruliferous treatments, 90%, 93%, and 97% of the whiteflies at 24, 48, and 72 hours, respectively, tested positive for TYLCV.

Select up- and downregulated genes from the RM and TQ DEG analyses were validated using a QIAcuity One digital PCR (dPCR) system (QIAGEN, Hilden, Germany) (Supplementary Table 1). Full-length coding sequences of each gene in the B. tabaci MEAM1 genome (version 1.2; Chen et al. 2016), downloaded at http://www.whiteflygenomics.org/ftp/MEAM1/v1.2/MEAM1_CDS_v1.2.fa.gz, were used to design primers with Primer3web74 (version 4.1.0) at default parameters (https://primer3.ut.ee/). Primers for each DEG are listed in Supplementary Table 2. dPCR Reactions consisted of 5 µL EvaGreen master mix (QIAGEN, Hilden, Germany), 0.6 µL 10 µM forward and reverse primers, 1.5 µL cDNA at 500 ng/µL, and 7.3 µL nanopure water. Reactions were performed in 24 or 96 well nanoplates with 8.5k partitions per well. Thermocycling consisted of an initial 2 min denaturation at 95 °C, 40 cycles at 95 °C for 30 s and 60 °C for 60 s, and a final extension for 10 min at 35 °C. Imaging was performed at an exposure duration of 200 ms and gain of 6, and the threshold for positive partitions was set at 50 RFU. The expression of each gene was normalized to the whitefly gene α-tubulin, a housekeeping gene.