ADVANCING HOST-PLANT RESISTANCE TO COTTON LEAFROLL DWARF VIRUS, AN EMERGING COTTON VIRAL DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1025409
• Grant No.: 2021-67028-34097
• Cumulative Award Amt.: $294,000.00
• Proposal No.: 2020-05210
• Project Start Date: Jun 1, 2021
• Project End Date: May 31, 2025
• Grant Year: 2021
• Program Code: [A1701]- Critical Agricultural Research and Extension: CARE

Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016

Performing Department
Crop and Soil Sciences

Non Technical Summary
The Cotton Leafroll Dwarf Virus, which causes Cotton Blue Disease (CBD), is regarded as the second most damaging viral disease to commercial cotton worldwide. The recent discovery of CLRDV in the U.S. indicates this virus may pose a significant threat to the profitability of cotton production. Identifying durable sources of genetic resistance and the underlying mechanisms that contribute to the trait is an important step in the development of resistant varieties. In this proposed research, a team of scientists, which include a breeder (Koebernick), geneticist (Chee), and virologist (Bag) with complementary skill sets, will work closely with an engaged stakeholder (Jones) to undertake activities salient to germplasm evaluation and pre-breeding for CLRDV resistance, and to the development and application of tools to predict phenotype from genotype to accelerate breeding of CLRDV resistant cultivars. The specific objectives of this project are to: 1)identify sources of resistance to CLRDV in Upland cotton, 2) genetic mapping to identify diagnostic markers for the resistant gene(s), and 3) elucidate and characterize putative defense genes against the reddening and leaf downward cupping symptoms that are commonly associated with CLRDV. We expect this project to uncover new scientific knowledge and genetic resources for genetic improvements of host plant-resistance for this emerging viral disease. This proposal is submitted in response to and supported by the Cotton Board and aims to develop and deploy germplasm, genetic, and molecular tools for resistance to CLRDV.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1710	1081	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1710 - Upland cotton;

Field Of Science
1081 - Breeding;

Keywords

blue disease

gbs

gossypium hirsutum

rnaseq

host-plant resistance

invasive virus

Goals / Objectives
This project addresses the development of a disease management solution by deepening our understanding of interactions between the U.S. cotton crop and its new viral pathogen. TheCotton Leafroll Dwarf Virus(CLRDV; genusPolerovirus, familyLuteoviridae),which causes Cotton Blue Disease,is regarded as the second most damaging viral disease to commercial cotton production worldwide. This viruswas first described in African and has since spread to major cotton-producing countries in South America and Asia. In 2018, numerous cotton fields across the Southeastern states were observed to have symptomatic plants resembling a viral disease, which was later confirmed to be infected with an RNA virus resembling CLRDV. Phylogenetic analyses have nowshown that the CLRDV isolates collected from the across U.S. cotton-belt are monophyletic and form a different clade from CLRDVisolatesfrom South America, suggesting that the U.S. virus is a novel genotype resulting from a single introduction that likely occurred recently. This newly introduced virus may pose a significant threat to U.S. cotton and highlight the need to identify management strategies to avert or mitigate the economic impact on cotton production. Deployment of resistant cultivars has been shown to be the most efficacious strategy for managing CLRDV in South America. However, resistance lines from Brazil were found to be susceptible to the U.S. isolate, suggesting that breeding for host-plant resistance will necessitate additional screening to identify new sources of resistance and DNA markers linked to the resistance genes. The goals of the project are to identify and release newly identified sources of resistance to CLRDVand todevelop an understanding of thegenetic mechanisms of host-plant resistanceand use breeding approaches to deliver grower-improved solutions (biomarkers) in the short and medium time scale.Identification of germplasm resistant to the virus will have an immediate impact on U.S. cotton breeding programs across the public and private sectors, while the knowledge generated on the genetics of resistance and the molecular breeding tools developed to expedite the development of resistant cultivars will ensure the economic and environmental viability of the cotton industry. The specific objectives of this project are to: 1) identify sources of resistance to CLRDV in Upland cotton, 2) genetic mapping to identify diagnostic markers for the resistant gene(s), and 3) elucidate and characterize putative defense genes against the reddening and leaf downward cupping symptoms that are commonly associated with CLRDV.Objective 1:Identify sources of resistance to CLRDV in Upland cotton.Preliminary screening conducted at two Alabama locations in 2019 showed that six percent of the 800 public germplasm accessions including wild race stocks, obsolete varieties, and elite breeding lines to be a non-host for the CLRDV virus via the presence or absence of symptoms and RT-PCR diagnostic assay. As a foundation for the project, we will validate the putative resistant lines to confirm the phenotype observed as well as evaluate for productivity and fiber quality. Additionally, we will screen an additional 200 elite breeding lines not tested in 2019.Objective 2:Genetic mapping to identify diagnostic markers for the resistant gene(s).Two complementary approaches will be utilized for marker-trait association analysis. First, a GWAS analysis will be conducted on approximately 1,000 lines currently being screened in Activity 1. Second, marker-trait association analysis will be conducted using already developed F2:3populations developed by crossing two resistant lines from diverse origins to a common susceptible parent from an elite background. Additionally, we will draw on populations that are segregating for a specific symptomology described in activity 3 (below).Objective 3:Elucidate and characterize putative defense genes against the reddening and leaf downward cupping symptoms that are commonly associated with CLRDV.In Alabama and Georgia, the most commonly observed symptoms of infected plants are the reddening and downward cupping/weathering of leaves in seedlings with mature plants also exhibiting leaf rolling and shortening of internodes. We have observed several F2populations in a breeding nursery segregating for these symptoms and have identified parents that are contributing to these phenotypes. We will conduct a comparative transcriptome profiling study via RNA sequencing on the three parental lines of populations segregating for leaf reddening and downward cupping symptoms as described in Objective 2 to identify differentially expressed genes in symptomatic and asymptomatic plants during CLRDV infection.By focusing on genes in the genomic region identified in Activity 2, we seek to identify putative candidates for the causal gene.

Project Methods
Objective 1:Screening will be conducted in the CLRDV disease nursery atFairhope and Talassee, AL,both areas that showed significant CLRDV presence in previous years.This trial will be a randomized complete block design with one row plots that are 10ft long, with two replications. The seed will have no insecticides treatment and will be planted in the first week of May; the trial will utilize no aphid management to ensure high insect populations. These trials will be monitored weekly for aphids and for virus symptoms. During each evaluation, the presence/absence of the different symptoms will be recorded. Plant growth-related symptomology that will be recorded includes leaf crinkling, cupping and thickening of leaves, reddening of leaf veins and petioles, shortened internodes, dwarfing of the plant, swollen and brittle stems, and accentuated verticality of top growth.In early fall, plant tissue from all plots will be collected in a plastic bag and transported in a cooler with ice to the diagnostic lab (Bag's lab). Samples will be stored in a refrigerator and tested for the virus using RT-PCR to confirm that the symptoms observed are associated with the virus.Cotton germplasm with no symptomatic plants and the RT-PCR diagnostic test on pooled RNA samples showing negative results in both testing locations would indicate resistance to the virus.A prerequisite of any field-screening of germplasm to identify sources of resistance is the availability of sufficient seed supply.Seed increase fields have been identified and will be available for our use both in the U.S. for the summer increase in Maricopa, Arizona, and in the Cotton Winter Nursery near Liberia, Costa Rica. Dr. Jones is the project manager for both the Cotton Summer and Winter Nursery and has ready access to it whenever the need arises.Objective 2:.For genotyping, approximately 100mg of wet-tissue will be harvested using standard leaf punches into a sealable deep-well plate containing silica beads. Genomic DNA will be extracted with an adapted procedure of John (1992), that includes modifications for automation. In brief, lyophilized leaf material will be pulverized into a fine powder using a GenoGrinder (Spex SamplePrep LLC) and immediately treated with lysis buffer containing 1% insoluble PVPP. Supernatant particulates will be removed by using a 96-well filter (Pall AcroPrep; Sigma Aldrich) by a robotic transfer.Purified gDNA will be quality checked and normalized. Genotyping by sequencing will be conducted according to the modified methods of Elshire et al., 2011 (Elshire et al. 2011). Briefly, a survey of restriction profiles will be performed on representative Upland cotton gDNA to select the best double-digest enzyme pairs (e.g.,MspI +PstI and others) for enriching the abundance of fragments in the 300bp - 700bp range. A pool of indexed sequencing libraries will be prepared and sequencing will be conducted on an Illumina NovaSeq using paired-end sequencing (2x150bp PE) to collect approximately 3M reads per individual. Raw sequence data will be purified of low-quality bases and adapter sequences with the reads will be aligned to the high-quality Upland cotton reference assembly v1.1 with the GMAP/GSNAP short read aligner (Wu et al. 2016). The resulting BAM alignment files will be sorted and indexed with Samtools (Li et al. 2009). Genotypes will be called using the Genome Analysis Tool KIT (GATK) Haplotype Caller (McKenna et al. 2010), which is an ideal tool for calling variants in polyploid genomes that have several alternate alleles. The raw VCF file will be carefully filtered to remove insertions/deletions (INDELS), and for read depth and an assessment of minor allele frequency ranges (with the assumption that the favorable alleles we are after are, in fact, the minor alleles). Data analysis of phenotypes will be performed by developing a linear model containing terms for the design factors and the population structure. Analysis of variance (ANOVA) techniques for mixed models (available in R, (R Core Team 2014)) will be used to analyze the model. The models will also be further developed to include markers to identify marker-trait associations. We will perform GWAS using MLM (Yu et al. 2006), which accounts for population structure and kinship that is implemented in Genome Association and Prediction Integrated Tool (GAPIT) (Lipka et al. 2012) and FarmCPU (Liu et al.). Model selection will be used to determine the optimal number of principal components based on Bayesian Information Criteria (Zhu and Yu 2009). For whole-genome shotgun sequencing of the resistant parents identified in Objective 1 (~10), we will prepare sequencing libraries following standard Illumina TruSeq procedures. Each parent used in the biparental population will be sequenced to an approximate 30X depth using a 2x150bp paired-end approach on an Illumina NovaSeq. Sequences will be filtered and aligned to the v1.1 reference assembly as described above and used in the GWAS analysis to define haplotype groups that underlie various forms of CLRDV resistance.Objective 3:.A total of 48 cDNA libraries will be developed, which include three replications each from the following four cotton cultivars with and without CLRDV infection and the four sampling dates.Two weeks old cotton seedlings will be used for inoculation by manually placing aphids that have acquired the virus from infected plants (Galbieri et al. 2017). Ten days after inoculation, the aphids are then eliminated by spraying insecticides. RNA will be collected from leaf tissues of each replicate for all cotton cultivars separately at 5, 10, 15, and 20 days after virus transmission by Dr Bag, which spanned the times at which the early symptoms and later-stage reactions were previously observed.cDNA library construction and high throughput RNA transcript sequencing will use methods described in Severin et al (2010).Briefly, cDNA library construction will use total RNA extracted from leaf tissues using the TruSeq RNA sample preparation kit, which employs polyA selection from mRNA enrichment.Sequencing will use an Illumina RNA Hi-Seq at the Georgia Genomics Facility.

Progress 06/01/23 to 05/31/24

Outputs
Target Audience:The target audience includes plant geneticists, pathologists, and breeders working on developing disease management solutions to the recently discovered cotton leafroll dwarf virus - CLRDV. The PD's have disseminated research findings to these research communities through publications of their research findings in refereed journals and presentations at scientific meetings and other venues. For example, a PhD student working in Dr Bag's lab has given a presentation in two research conferences. Also, Co-PD Dr. Koebernic is a regular participant in the monthly meetings of the CLRDV Working Group, which is organized by the USDA-ARS and moderated by Dr. Tim Widmer. Members of this working group include entomologists, weed scientists, virologists, geneticists, and plant disease diagnosticians. The discussion focuses on sharing results from ongoing projects and identifying challenges and opportunities for future research. Additional audiences who have benefited from this research include members engaging in the cotton value chain such as producers, processors, and consumers. The PDs regularly interacts with cotton producers by participating in field-day activities. For example, both PIs Chee and Bag gave presentations on topics related to the symptomology and potential impact of the CLRDV virus on cotton production during the Cotton Commission Research Review day, which was held at Midville, GA on July 25thof 2023. More than 30 cotton growers from across Georgia attended the meeting, including Director of the Georgia Cotton Commodity Commission, Mr. Taylor Sills, and other board members. Also in attendance was co-PD Dr. Jones from Cotton Incorporated. Changes/Problems:Compared to the 2022 field season, which experienced heat wave in early July resulting in widespread expression of viral symptoms in susceptible genotype, the summer season of 2023 was much milder and wetter. Consequently, the expression of symptoms from known susceptible genotypes was sporadic and inconsistent. We conducted a second year of disease screening on the recombinant inbred population known to be segregating for the putative QTL region for the susceptible trait. The population consisted of 280 F6 lines from the cross of U1 by GA16016, and was planted in 3 replications at the UGA Gibbs Farm near Tifton, GA. It is possible that the 2023 weather pattern was not conducive to symptoms expression, as the segregation for disease incidence was not consistent with the 2022 season. Additionally, some adult plants exhibited symptoms similar to CLRDV but were later diagnosed by plant pathologists as water logging, which may have affected our scoring accuracy. Therefore, we plan to repeat this screening experiment in the 2024 growing season and have requested a 12-months no cost extension, which would provide another growing season to complete the project. What opportunities for training and professional development has the project provided?Two graduate students, Bukhtaweer Talat and Surendra Edula, were integral to this project. A postdoctoral scientist, Dr Iram Raza, and a graduate student, Iago Schardong, have assisted with genetic mapping and bioinformatic data analysis. Finally, field technicians Dr. Ed Lubbers and Jennifer McBlanchett were instrumental in planting, data collection, and plant tissue sampling for DNA/RNA analysis. An undergraduate, Blake West, also assisted in the project. How have the results been disseminated to communities of interest?Graduate student, Surendra Edula, has presented a talk in two research conferences relating to plant pathology. Edula, R.S., Hand, L.C., Snider, J.L, Chee, P.W., Kemerait, R.C., Roberts, P.M., and Bag, S. Characterization of Caulimovirid-like sequences in upland cotton (Gossypium hirsutumL.) in Georgia, USA. 3-PEAT GAPP Meeting, Savannah, GA, March 5-6, 2024. Reddy, S.E., Bag, S., Milner, H., Kumar, M., Suassuna N.D., Kemerait, R.C., Chee, P.W., Hand, L.C., Snider, J.L., Srinivasan, R., and Roberts, P.M.Aphid-transmitted cotton leafroll dwarf virus and host response in Georgia. Entomological Society of America 2023 Annual Conference, National Harbor, Maryland, November 5-8, 2023. What do you plan to do during the next reporting period to accomplish the goals?Results from the 2023 growing season provided inadequate information on the segregation of CLRDV symptoms in the recombinant inbred population and the F3 progeny testing. An additional growing season is needed to validate the phenotype observed in the 2022 and 2023 field seasons and to genotype the F3 populations.A 12-months no-cost extension will cover the additional phenotypic data collection and allow additional time for analyzing the marker-trait association, the RNASeq dataset, and drafting manuscripts for publication.

Impacts
What was accomplished under these goals? Progress is being made on all three activities listed in the proposed research. Activity 1; Germplasm screening and phenotyping. Screening of diverse cotton germplasm have been completed and a manuscript describing the results have been submitted to a refereed journal. Also, a manuscript focusing on the symptomology of CLRDV is mostly completeActivity 2; Genetic mapping to identify diagnostic markers for the resistant genes. A graduate student working on identifying diagnostic markers for the resistant genes to CLRDV has identified a putative recessive gene for the resistance trait. To validate this result, a recombinant inbred population was screened twice in the funding period (2022 and 2023) but the results was inconsistent between years.Additionally, a bulk symptomatic and asymptomatic F3 plants for QTLSeq analysis has been developed. Activity 3; Elucidate and characterize putative defense genes against the symptoms of reddening and downward-cupping of the leaves. Transcriptomic sequencing of CLRDV resistant and susceptible lines has been completed, and data analysis is ongoing as of the end of the reporting period.

Publications

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:The target audience includes geneticists, pathologists, and breeders working on developing disease management solutions to the recently discovered cotton leafroll dwarf virus - CLRDV. The PI's are well poised to address these research communities through publications of their research findings in refereed journals and presentations at scientific meetings and other venues. For example, co-PI Dr Bag has presented his research findings in a talk titled "Cotton leafroll dwarf disease: an enigmatic virus disease on cotton in Georgia, USA" at the 15thInternational Symposium of Plant Virus Epidemiology Conference, which was held in Madrid, Spain. He also presented a similar talk at the ESA-Southeastern Branch and APS-Caribbean Division Joint Meeting, which was held in Puerto Rico. Additional clienteles who have benefited from this research include members engaging in the cotton value chain such as producers, processors, and consumers. The co-PI Dr Jones specifically, but also the other PIs, regularly interacts with cotton producers by participating in field-day activities. For example, both PIs Chee and Bag gave presentations on topics relating to the symptomology and potential impact of the CLRDV virus on cotton production during the Cotton Commission Research Review day and the Cotton and Peanut Research field day, which was held at Tifton GA on July 29 and September 7 of 2022, respectively. In the later meeting, over 100 growers from Georgia and surrounding states were in attendance.Finally, co-PI Dr Bag presented a talk at the UGA Cotton Production Workshop, which was held at Tifton, GA on January 25, 2023. Changes/Problems:The heat wave in early July of 2022 have resulted in more widespread expression of viral symptoms in susceptible genotype. An inbred population known to be segregating for the putative QTL region for the susceptible trait was planted in 3 replications at the UGA Gibbs Farm near Tifton, GA, and the segregation for disease incidence was collected. This population have also been genotyped using GBS markers. Additionally, we observed several F3 populations were segregating for susceptibility and several bulk DNA samples from about 30 symptomatic and asymptomatic lines were collected for QTLSeq analysis. Sequencing data was received in late April and data analysis in progress. What opportunities for training and professional development has the project provided?Two graduate students, Bukhtaweer Talat and Surendra Edula, were integral to this project. A postdoctoral scientist, Dr Iram Raza, and a graduate student, Iago Schardong, have assisted with genetic mapping and bioinformatic data analysis. Finally, field technicians Dr. Ed Lubbers and Jennifer McBlanchett were instrumental in planting and collecting tissue samples for DNA/RNA analysis. How have the results been disseminated to communities of interest?Two presentations in International Conference, several in professional societal annual meetings and cotton growers workshop were given on CLRDV. Also, anarticle was published in a refereed journal. What do you plan to do during the next reporting period to accomplish the goals?Perform association analysis using the phenotypic data collected from the inbred population, using the GBS data. In addition, we will analyze the QTLSeq dataset to supplement the biparental population data to identify QTL for the resistant phenotype. Also, we replant the progenies used in the QTL analysis to validate the phenotype observed in the 2022 field season. Finally, we will use the markers linked to the susceptible/resistant phenotype to predict plants that will show symptoms, tracking their disease progression and perform RNASeq analysis to identify genes that are up or down regulated during the onset of disease.

Impacts
What was accomplished under these goals? Progress is being made on all three activities listed in the proposed research. Activity 1; Germplasm screening and phenotyping. Screening of diverse cotton germplasm have been completed. Activity 2; Genetic mapping to identify diagnostic markers for the resistant genes. A graduate student has been working on identifying diagnostic markers for the genes that confer resistance to CLRDV. Two breeding populations previously identified to be segregating for CLRDV symptoms have been genotyped using theCotton SNP63K Illumina arrayand the SNP dataset is being analyzed to determine the markers-trait associations (Objective 2.1b). An additional recombinant inbred population has been phenotyped and a bulk symptomatic and asymptomatic F3 plants for QTLSeq analysis has been developed. Activity 3; Elucidate and characterize putative defense genes against the symptoms of reddening and downward-cupping of the leaves. Transcriptomic sequencing of CLRDV resistant and susceptible lines has been completed and the data analysis will commence once the student has been trained in bioinformatics and is familiar with the data-processing pipeline.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Surendra R. Edula, Sudeep Bag, Hayley Milner, Manish Kumar, Nelson D. Suassuana, Peng W. Chee, Robert C. Kemerait, Lavesta C. Hand, John L. Snider, Rajagopalbabu Srinivasan, Phillip M. Robert. 2023. Cotton leaf dward disease: An enigmatic viral disease in cotton. Molecular Plant Pathology. DOI:10.1111/mpp.13335

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:The target audience includes geneticists, pathologists, and breeders working on developing disease management solutions to the recently discovered cotton leafroll dwarf virus - CLRDV. The PI's are well poised to address these research communities through publications of their research findings in refereed journals and presentations at scientific meetings and other venues. For example, a graduate student in the co-PI's (Dr Bag) lab has presented his research findings in the Crop Science Society of America Annual Meeting in Salt Lake City, UT, and in the PI's lab at the Beltwide Cotton Conference, which was held virtually. Additional clienteles who have benefited from this research include members engaging in the cotton value chain such as producers, processors, and consumers. The co-PI Dr Jones specifically, but also the other PIs, regularly interacts with cotton producers by participating in field-day activities. For example, both PIs Chee and Bag gave presentations on topics relating to the symptomology and potential impact of the CLRDV virus on cotton production during the Cotton Commission Research Review day and the Cotton and Peanut Research field day, which was held at Tifton GA on July and September of 2021, respectively. Changes/Problems:The 2021 growing season was not conducive to the expression of viral symptoms due to mild temperature and excessive rain in July and August, thus hampering our ability to perform progeny testing in segregating populations. An inbred population will be utilized this year, which will allow us to replicate the genotypes in phenotyping. Provide a higher density marker coverage for a small subset of segregating populations to map and fine-map QTLs by supplementing GBS markers with the Cotton SNP63K Illumina array. What opportunities for training and professional development has the project provided?Two graduate students, Divya Sharma and Afsha Tabassum, were integral to this project. A postdoctoral scientist, Dr Sameer Khanal, has assisted with genetic mapping and other data analysis. Finally, field technicians Dr. Ed Lubbers and Jennifer McBlanchett were instrumental in planting and collecting tissue samples for DNA/RNA analysis How have the results been disseminated to communities of interest?Two presentations were presented in professional societal annual meetings. In addition, two manuscripts were accepted for being published in PLOSOne and Frontiers in Plant Science What do you plan to do during the next reporting period to accomplish the goals?Utilizing an inbred population, which allows us to replicate each genotype for disease screening, would provide more reliable phenotypic data. Supplementing GBS markers with those from the Cotton SNP63K Illumina array for a small subset of segregating populations would provide a higher density of markers needed to map and fine-map the QTLs for the resistance genes.

Impacts
What was accomplished under these goals? Progress is being made on all three activities listed in the proposed research. Activity 1; Germplasm screening and phenotyping. A subset of the 800 cotton germplasm lines initially screened for reaction to CLRDV were re-evaluated in a field in Tallassee, Alabama. Tissue samples were collected from each genotype and DNA extraction is in progress. Activity 2; Genetic mapping to identify diagnostic markers for the resistant genes. A graduate student has been assigned to this project, whose current efforts are focusing on genetic mapping to identify the diagnostic markers for the genes that confer resistance to CLRDV. Two breeding populations previously identified to be segregating for CLRDV symptoms have been genotyped using theCotton SNP63K Illumina arrayand the SNP dataset are being analyzed to determine the markers-trait associations (Objective 2.1b). Activity 3; Elucidate and characterize putative defense genes against the symptoms of reddening and downward-cupping of the leaves . Transcriptomic sequencing of CLRDV resistant and susceptible lines have been completed and the data analysis will commence once the student has been trained in bioinformatics and is familiar with the data-processing pipeline.?

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Parkash, V., Sharma, D. B., Snider, J., Bag, S., Roberts, P., Tabassum, A., . . . Chee, P. (2021). Effect of Cotton Leafroll Dwarf Virus on Physiological Processes and Yield of Individual Cotton Plants. FRONTIERS IN PLANT SCIENCE, 12, 13 pages. doi:10.3389/fpls.2021.734386 Tabassum, A., Bag, S., Suassuna, N. D., Conner, K. N., Chee, P., Kemerait, R. C., & Roberts, P. (2021). Genome analysis of cotton leafroll dwarf virus reveals variability in the silencing suppressor protein, genotypes and genomic recombinants in the USA. PLOS ONE, 16(7), 17 pages. doi:10.1371/journal.pone.0252523