Brief Summary of Minutes of Annual Meeting

The soybean virus symposium titled Mechanisms of Resistance to Soybean Viruses was held on October 10, 2009 from 10 AM to 5 PM. The symposium started with opening remarks from Drs. David Wright (NCERA President, 2009) and John Hill. John Hill told the group that David Wright received the prestigious Henry Wallace Distinguished Award from Iowa State University in 2009. There were a total of four talks presented: (1) Mechanisms of Resistance of Plants to Pathogens: Are common mechanisms or case-by-case mechanisms more relevant? Presenter: Andrew Bent, University of Wisconsin, (2) Driving Innate Immunity: Emerging perspectives on the role of the ER, nucleus, and chloroplasts Presenter: S. Dinesh-Kumar, Yale University, (3) Rsv1/soybean mosaic virus research Presenters: Saghai Maroof, Virginia Polytechnic Institute and Raza Hajimorad, Univ. of Tennessee, (4) Virus induced gene silencing (VIGS) for gene function studies in plants - presenter: S. Dinesh-Kumar, Yale University.

The symposium was adjourned at 5 PM after a15 minutes open discussion and comments at the end.

The annual business meeting started at 8:30 AM on October 11. Attendees: David Wright (NCSRP), Rouf Mian (USDA-ARS, Ohio), Aoedra Kachroo (University of Kentucky), Sue Tolin (Virginia Tech), Saghai Maroof (Virginia Tech), Reza Hajimorad (University of Tennessee), Sead Sabanadzovic (Mississippi State), Ioannis Tzanetakis (Arkansas), Al Eggenberger (Iowa State University), Feng Qu (Ohio State University), Steven Slack (Ohio State University), John Hill (Iowa State University), Houston Hobbs (University of Illinois), Curt Hill (University of Illinois), Les Domier (USDA-ARS, Illinois), Steve Whitham (Iowa State University)

President, David Wright, welcomed the group and presented a brief overview of the program. The minutes from the 2008 annual meeting were discussed and approved unanimously.

Dr. Steve Slack welcomed the group and requested that every one should review the existing participant list for his/her own institute and update the list to reflect the current situation.

The state reports were presented by one or more representatives as follows:

Iowa: by John Hill and Steve Whitham; Illinois report by Les Domier; Arkansas by Ioannis Tzanetakis; Mississippi by Sead Sabanadzovic; Tennessee by Reza Hajimorad; Kentucky by Aardra Kachroo; Virginia by Sue Tolin; and Ohio by Rouf Mian and Feng Qu. Please see reports from states below.

Dr. Wright requested that every state send the electronic copy of their report to him and Rouf by the end of November. He also requested other state representatives who were not present at the meeting to send their report electronically so that those can be included in the annual report.

Nomination for the post of secretary for was sought. Rouf Mian nominated Reza Hajimorad for the post and John Hill seconded. No other nomination was made and Dr. Hajimorad was unanimously selected as the secretary for 2010.

Following the tradition, Rouf Mian, Secretary for 2009, became the President for 2010. Drs. Slack, Rouf Mian and others in the meeting expressed their thanks to Dr. Wright for his outstanding job as the 2009 President.

The following items for 2010 were discussed:

1. Nov 9-10 or Nov 16-17 was proposed as possible dates for the NCERA 200 Symposium for 2010. One of the two dates will be confirmed later after learning from all participants.

2. Tentative organizing committee for 2010: Drs. John Hill, David Wright, Reza Hajimorad, and Rouf Mian

3. Tentative proposal writing committee: Drs. Les Domier, David Wright, Steve Whitham, Reza Hajimorad, Sue Tolin, and Rouf Mian

State Reports 2009:

Illinois: Investigators: Leslie L. Domier, Glen L. Hartman, Curtis B. Hill, Houston A. Hobbs, Nancy K. McCoppin, Thanuja Thekke Veetil, Sushma Jossey

Virus distribution and incidence: Houston Hobbs worked with scouts of Asian soybean rust sentinel plots in Illinois to collect samples that were used to estimate the incidence of soybean-infecting viruses in Illinois. In 2008, Bean pod mottle virus (BPMV) was the most prevalent virus and was detected in 10 of 28 sentinel plots. In 2009, commercial soybean fields in seven counties in southern Illinois were assayed for virus infection. BPMV was detected in four of seven counties and Soybean dwarf virus (SbDV) was detected in one, Fayette County in south central Illinois. With this finding, SbDV has been detected in commercial soybean fields in nine Illinois counties.

Identification and characterization of Soybean yellow mottle mosaic virus: A new soybean infecting virus, Soybean yellow mottle mosaic virus (SYMMV), was identified in Korea. SYMMV initially induces bright yellow mosaic symptoms on leaves followed by stunting and reduced growth of older leaves. SYMMV is most similar to, but distinct from the Carmovirus Cowpea mottle virus, which is widespread in West Africa, and is transmitted by beetles and through seed. To determine whether SYMMV is present in the United States, quantitative RT-PCR assays were designed and used to assay soybean plants from Illinois and Mississippi for SYMMV infection. SYMMV was detected in six of ten 100-leaf pools from a research field in Stoneville, Mississippi that contained 19 soybean germplasm lines (including five from Korea). The nucleotide sequence of the 4009 nt genomic RNA of the Mississippi isolate of SYMMV (FJ707484) was 96% identical to the Korean SYMMV isolate. Because of the sampling techniques used, it was not possible to associate SYMMV-positive plants with disease symptoms.

Evaluation of Commercial Varieties for Soybean mosaic virus (SMV) Resistance: SMV is one of the most damaging soybean-infecting viruses worldwide. To provide soybean growers information about the responses of commercial soybean varieties to SMV infection, Houston Hobbs rated about 375 varieties for their responses to SMV infection in 2009. In the first two rounds of screening, seedlings were inoculated at the unifoliate stage with SMV G1 and varieties with symptoms were eliminated. In the final round of screening, 4-6 plants of each cultivar were inoculated with SMV G1 and screened for virus infection by ELISA. Seven soybean cultivars (Beck 376 NL, Merschman Truman 938LL, Merschman Wilson 1037LL, Merschman Miami 949LL, Nutech 3378L, Horizon 36-66L, and UniSouth Genetics USG 5601T) were identified as resistant to SMV G1. This is an increase over last year when just one of 350 varieties was resistant to SMV G1. The responses of all varieties were posted on the Varietal Information Program for Soybeans (VIPS) website (www.vipsoybeans.org)

Functional genomics of soybean seed development: In collaboration with Said Ghabrial at the University of Kentucky, Sushma Jossey, Ajay Singh, and Nancy McCoppin constructed a series of potential vectors for virus induced gene silencing (VIGS) based on Tobacco streak virus (TSV). Multicloning sites (MCSs) were inserted into full-length clones of RNA2 at three locations downstream of the 2B gene, a duplicated subgenomic promoter and MCS into the intergenic region of RNA3, and Thosea asigna virus CHYSEL sequence and MCSs into RNA3 upstream of the 3A coding region and upstream and downstream of the coat protein (3B) coding region. Soybean seedlings were biolistically inoculated with cDNAs of wild-type and modified viruses. All of the modified clones were infectious. All but one containing a deletion in the 2B gene produced symptoms indistinguishable from the unmodified virus. Sequencing representing a portion of the phytoene desaturase gene and a complete green fluorescent protein gene were inserted into the modified viruses. The modified viruses tested so far have not retained inserted sequences in systemic leaves. To attempt to stabilize the inserts, alternative insertion locations are being evaluated and a full-length clone of RNA1 from a divergent TSV isolate from Kentucky is being substituted for the cognate Illinois RNA1 clone.

A source of resistance to Asian soybean aphids: Asian soybean aphids (Aphis glycines) are major insect pests of soybean. Previously Curt Hill, Brian Diers, and Glen Hartman identified and mapped two dominant genes, Rag1 and Rag2, from "Dowling" and "Jackson", respectively, that confer resistance to soybean aphids. Plant introduction (PI) 200538 also shows strong resistance to soybean aphids. To determine the inheritance of resistance and to map gene(s) controlling resistance in PI 200538, F2 populations were developed from crosses between PI 200538 and three susceptible genotypes ("Ina", LD02-4485 and "Williams 82") and assayed for segregation of resistance and microsatellite markers. In all three crosses, resistance to soybean aphids segregated as a single dominant gene in a 3:1 genetic ratio. Segregation among F2:3 families from the crosses confirmed the dominant resistance gene hypothesis. The gene mapped to soybean linkage group F, flanked by the microsatellite markers Satt510, Soyhsp176, Satt114, and Sct_033, located in the same region as Rag2. Since the resistance gene in PI 200538 also gave resistance to soybean aphid biotypes 1 and 2, it is possible that the gene is Rag2 and not a new aphid resistance gene. Therefore, PI 200538 may be an additional source of Rag2.

Genetics of SMV seed transmission: Seed-borne infections are the primary sources of inoculum for SMV infections in the Midwest. To identify regions of the soybean genome that permit SMV to be transmitted efficiently through seed, populations of recombinant inbred (RI) lines were generated and phenotyped in replicated trials for transmission of SMV through seed. Seed transmission rates of SMV ranged from 0 to 43% among the lines. Nancy McCoppin scored 120 microsatellite markers in 250 soybean RI lines to map soybean genes involved in seed transmission of SMV and seed-coat mottling. She also scored seeds from each population for the color of mottling, the percent of mottled seed and the degree to which seed coats were mottled. Next, the molecular and phenotypic data will be combined and analyzed.

Sources of resistance to Tobacco streak virus: In soybean, TSV can cause significant yield losses and induces symptoms ranging from mild mosaic to bud blight, pod necrosis and plant death depending on the virus strain, host genotype and plant age at the time of infection. TSV also is transmitted through seed at rates ranging from 0 to >90% depending on the soybean line and virus isolate. To identify sources of resistance to TSV, Houston Hobbs is analyzing Ancestral and Diverse Sets of the USDA soybean germplasm collection for their responses to TSV infection. The Ancestral Set contains 95 lines that represented 99% of the genetic variability of public soybean cultivars as of the 1980's. To date, 92 of 95 Ancestral Set lines have been tested and are susceptible to TSV. The Diverse Set, which contains 3000 soybean lines, was assembled by Dr. R. Nelson, Curator of the Soybean Germplasm Collection, and represents genetic diversity not necessarily covered by the Ancestral Set. From the first 1000 lines in the Diverse Set, nine soybean lines were identified that appeared to be resistant to infection by an Illinois soybean isolate of TSV after multiple rounds of inoculation of plants from each line. Additional testing of the lines will be necessary to verify resistance and determine whether resistance, if verified, is general or specific to this isolate of TSV. Tanner, a minor contributor to the genetic diversity of modern soybean lines, was earlier shown in our research to be resistant to TSV.

Soybean dwarf virus diversity: SbDV is obligately transmitted by colonizing aphids in a persistent manner. Thanuja Thekke Veetil analyzed the amino acid sequence diversity of readthrough proteins (RTPs) of 24 dwarfing isolates of SbDV from Wisconsin and Illinois. The RTP, a minor component of viral capsids, has a significant role in specificity of aphid transmission of members of the Luteoviridae. Among the isolates, nucleotide sequence identities ranged from 95 to 100%. The predicted amino acid sequences differed at 56 positions in the 54-kDa readthrough domain compared to only five positions in the 22-kDa core coat protein. Phylogenetic analysis of both predicted amino acid and nucleotide sequences showed three distinct clusters of SbDV isolates.

The goals of the project are to (1) enhance interaction among scientists in the North Central region who are engaged in fundamental and applied soybean virus research and (2) establish media for effective dissemination and communication of information about the incidence, identification, and management of soybean virus diseases in the North Central region.

The main accomplishments are: Screening for genetic resistance against viruses, Construction of a DNA-based virus induced gene silencing system for functional genomics of soybean seed development, Development of high-throughput DNA-based gene silencing technology for soybeans were funded by the North Central Soybean Research Program and the United Soybean Board and Exploring new resistance resources for threatening soybean diseases funded by the Iowa Soybean Association.

The group is working on several soybean virus diseases including bean pod mottle, soybean mosaic, alfalfa mosaic, and soybean dwarf. Much work is being conducted on bean pod mottle virus with grants from the North Central Soybean Research Program, United Soybean Board and the Iowa Soybean Association.

A major focus of the soybean virus research at the University of Kentucky (UK) is to utilize the novel bean pod mottle virus (BPMV)-based vector, developed at UK, for gene function studies and expression of valuable proteins in soybean. VIGS is especially useful for plants, such as soybean, that are recalcitrant to transformation. Emphasis is placed on identification of resistance genes to major soybean pathogens and on screening candidate antifungal proteins from diverse sources.

Iowa State University also has been using virus-induced gene silencing (VIGS) as a reverse genetics tool to study functions of specific plant genes. Because BPMV has been shown to be an effective VIGS vector for soybean, they have developed a unique DNA-based BPMV vectors to increase the efficiency and utility of VIGS for soybean functional genomics.

The University of Illinois reports Highly effective VIGS vectors have been developed for soybean based on BPMV, but BPMV rarely invades meristems or developing embryos. In contrast, TSV readily invades both of these tissues. Therefore, a VIGS vector based on [tobacco streak virus] TSV would permit the analysis of gene function in tissue types and developmental stages that would be difficult to affect using BPMV vectors.

In regard to soybean dwarf virus, researchers at the University of Illinois found that the efficiencies of transmission by A. glycines of SbDV isolates with variant coat protein sequences were compared and found to be very similar.

Wisconsin reports that there has been an increase in incidence of Alfalfa mosaic virus (AMV) in some of the soybean growing areas of the Northern states in recent years. However, absence of a fast, reliable, sensitive and easy diagnostic assay for AMV is not available. The research team attempted to develop a desirable monoclonal antibody to capable of detecting all AMV strains. Characterization of the produced antibodies showed that all belong to IgM sub-class; a sub-class not user friendly for assay development. None of these antibodies showed any satisfactory result in either of the assays.

Because of the recent increase in aphid and bean leaf beetle activity in North Dakota, another virus survey was conducted in July and August of 2007. Leaves were collected from 139 soybean fields in southeastern North Dakota and evaluated for presence of soybean mosaic virus (SMV) and bean pod mottle virus (BPMV) using Agdia virus kits. Nineteen fields tested positive for SMV and 8 were positive for BPMV. Repeated testing of leaf samples however, gave variable results with the Agdia kits.

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