Genetic characteristics of two genes for resistance to soybean mosaic virus in PI486355 soybean

Soybean [Glycine max (L.) Merr.] PI486355 is resistant to all the identified strains of soybean mosaic virus (SMV) and possesses two independently inherited resistance genes. To characterize the two genes, PI486355 was crossed with the susceptible cultivars Lee 68 and Essex and with cultivars Ogden and Marshall, which are resistant to SMV-G1 but systemically necrotic to SMV-G7. The F₂ populations and F₂₃ progenies from these crosses were inoculated with SMV-G7 in the greenhouse. The two resistance genes were separated in two F₃₄ lines, LR1 and LR2, derived from Essex x PI486355. F₁ individuals from the crosses of LR1 and LR2 with Lee 68, Ogden, and York were tested with SMV-G7 in the greenhouse; the F₂ populations were tested with SMV-G1 and G7. The results revealed that expression of the gene in LR1 is gene-dosage dependent, with the homozygotes conferring resistance but the heterozygotes showing systemic necrosis to SMV-G7. This gene was shown to be an allele of the Rsv1 locus and was designated as Rsv1-s. It is the only allele identified so far at the Rsv1 locus which confers resistance to SMV-G7. Rsv1-s also confers resistance to SMV-G1 through G4, but results in systemic necrosis with SMV-G5 and G6. The gene in LR2 confers resistance to strains SMV-G1 through G7 and exhibits complete dominance. It appears to be epistatic to genes at the Rsv1 locus, inhibiting the expression of the systemic necrosis conditioned by the Rsv1 alleles. SMV-G7 induced a pin-point necrotic reaction on the inoculated primary leaves in LR1 but not in LR2. The unique genetic features of the two resistance genes from PI486355 will facilitate their proper use and identification in breeding and contribute to a better understanding of the interaction of SMV strains with soybean resistance genes.     

Pyramiding multiple genes for resistance to soybean mosaic virus in soybean using molecular markers

Seven strains of Soybean mosaic virus (SMV) and three independent resistance loci (Rsv1, Rsv3, and Rsv4) have been identified in soybean. The objective of this research was to pyramid Rsv1, Rsv3, and Rsv4 for SMV resistance using molecular markers. J05 carrying Rsv1 and Rsv3 and V94-5152 carrying Rsv4 were used as the donor parents for gene pyramiding. A series of F_2:3, F_3:4, and F_4:5 lines derived from J05 × V94-5152 were developed for selecting individuals carrying all three genes. Eight PCR-based markers linked to the three SMV resistance genes were used for marker-assisted selection. Two SSR markers (Sat_154 and Satt510) and one gene-specific marker (Rsv1-f/r) were used for selecting plants containing Rsv1; Satt560 and Satt063 for Rsv3; and Satt266, AI856415, and AI856415-g for Rsv4. Five F_4:5 lines were homozygous for all eight marker alleles and presumably carry all three SMV resistance genes that would potentially provide multiple and durable resistance to SMV.     

Extreme resistance to cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins

For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomato plants were transformed with a combination of two coat protein (CP) genes, representing both subgroups of CMV. The CP genes were cloned from the CMV-D strain and Italian CMV isolates (CMV-22 of subgroup I and CMV-PG of subgroup II) which have been shown to produce severe disease symptoms. Four plant transformation vectors were constructed: pMON18774 and pMON18775 (CMV-D CP), pMON18831 (CMV-PG CP) and pMON18833 (CMV-22 CP and CMV-PG CP). Transformed R0 plants were produced and lines were selected based on the combination of three traits: CMV CP expression at the R0 stage, resistance to CMV (subgroup I and/or II) infection in growth chamber tests in R1 expressing plants, and single transgene copy, based on R1 segregation. The results indicate that all four vector constructs generated plants with extremely high resistant to CMV infection. The single and double gene vector construct produced plants with broad resistance against strains of CMV from both subgroups I and II at high frequency. The engineered resistance is of practical value and will be applied for major Italian tomato varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene

Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat‐growing areas in China. Because it is vectored by the fungus‐like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co‐transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12‐1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12‐1 showed broad‐spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild‐type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus‐derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad‐spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV.     

RSC3K of soybean cv. Kefeng No.1 confers resistance to soybean mosaic virus by interacting with the viral protein P3

Soybean mosaic virus (SMV) is one of the most devastating viral pathogens of soybean (Glycine max (L.) Merr). In total, 22 Chinese SMV strains (SC1–SC22) have been classified based on the responses of 10 soybean cultivars to these pathogens. However, although several SMV-resistance loci in soybean have been identified, no gene conferring SMV resistance in the resistant soybean cultivar (cv.) Kefeng No.1 has been cloned and verified. Here, using F₂-derived F₃ (F_2:3) and recombinant inbred line (RIL) populations from a cross between Kefeng No.1 and susceptible soybean cv. Nannong 1138-2, we localized the gene in Kefeng No.1 that mediated resistance to SMV-SC3 strain to a 90-kb interval on chromosome 2. To study the functions of candidate genes in this interval, we performed Bean pod mottle virus (BPMV)-induced gene silencing (VIGS). We identified a recombinant gene (which we named R_SC3K) harboring an internal deletion of a genomic DNA fragment partially flanking the LOC100526921 and LOC100812666 reference genes as the SMV-SC3 resistance gene. By shuffling genes between infectious SMV DNA clones based on the avirulent isolate SC3 and virulent isolate 1129, we determined that the viral protein P3 is the avirulence determinant mediating SMV-SC3 resistance on Kefeng No.1. P3 interacts with RNase proteins encoded by R_SC3K, LOC100526921, and LOC100812666. The recombinant R_SC3K conveys much higher anti-SMV activity than LOC100526921 and LOC100812666, although those two genes also encode proteins that inhibit SMV accumulation, as revealed by gene silencing in a susceptible cultivar and by overexpression in Nicotiana benthamiana. These findings demonstrate that R_SC3K mediates the resistance of Kefeng No.1 to SMV-SC3 and that SMV resistance of soybean is determined by the antiviral activity of RNase proteins.     

Natural resistance to cucumber mosaic virus in lupin species

Ten species of lupins (Lupinus spp.) were tested for resistance to cucumber mosaic cucumovirus (CMV) in field experiments where inoculation was by naturally-occurring aphid vectors, and in the glasshouse by sap or graft-inoculation. L. albus and six species of ‘rough-seeded’ lupins did not become infected with CMV either under intense inoculum pressure in the field or when graft-inoculated. Two L. hispanicus, 17 L. luteus and four L. mutabilis genotypes became infected with CMV in the field, but no infection was detected in L. hispanicus P26858 or seven L. luteus genotypes. CMV was detected at seed transmission rates of 0.2–16% in seedlings of infected L. luteus, differences in levels of seed transmission between genotypes being significant and relatively stable from year to year. Graft-inoculation of CMV to plants of six genotypes of L. luteus in which no infection was found in the field induced a systemic necrotic reaction suggesting that the resistance they carry is due to hypersensitivity. In L. hispanicus accessions P26849, P26853 and P26858, CMV sub-group II isolate SN caused necrotic spots in inoculated leaves without systemic movement, while sub-group I isolate SL infected them systemically without necrosis. Another sub-group I and two other sub-group II isolates behaved like SL in P26849 and P26853 but infected only inoculated leaves of P26858. This suggests that two strain specific hypersensitive resistance specificities are operating against CMV in L. hispanicus. When plants of L. luteus genotypes that gave hypersensitive reactions on graft-inoculation were inoculated with infective sap containing two sub-group I and seven sub-group II isolates, they all responded like L. hispanicus P26858. A strain group concept is proposed for CMV in lupins based on the two hypersensitive specificities found: strain group 1 represented by isolate SN which induces hypersensitivity with both specificities, strain group 2 represented by the three isolates which induced hypersensitivity only with the specificity present in L. luteus and L. hispanicus P26858, strain group 3 by as yet hypothetical isolates that induce hypersensitivity only in presence of the specificity in L. hispanicus P26849 and P26853 that responded just to isolate SN, and strain group 4 by isolate SL which overcomes both specificities. When F₂ progeny plants from crosses between hypersensitive and susceptible L. luteus parents were inoculated with isolate SN, the resistance segregated with a 3:1 ratio (hypersensitive:susceptible), suggesting that a single dominant hypersensitivity gene, Ncm-1, is responsible. As gene Ncm-1 had broad specificity and was not overcome by any of the five CMV isolates from lupins tested, it is valuable for use in breeding CMV resistant L. luteus cultivars.     

Natural resistance to cucumber mosaic virus in lupin species

Ten species of lupins (Lupinus spp.) were tested for resistance to cucumber mosaic cucumovirus (CMV) in field experiments where inoculation was by naturally-occurring aphid vectors, and in the glasshouse by sap or graft-inoculation. L. albus and six species of ‘rough-seeded’ lupins did not become infected with CMV either under intense inoculum pressure in the field or when graft-inoculated. Two L. hispanicus, 17 L. luteus and four L. mutabilis genotypes became infected with CMV in the field, but no infection was detected in L. hispanicus P26858 or seven L. luteus genotypes. CMV was detected at seed transmission rates of 0.2–16% in seedlings of infected L. luteus, differences in levels of seed transmission between genotypes being significant and relatively stable from year to year. Graft-inoculation of CMV to plants of six genotypes of L. luteus in which no infection was found in the field induced a systemic necrotic reaction suggesting that the resistance they carry is due to hypersensitivity. In L. hispanicus accessions P26849, P26853 and P26858, CMV sub-group II isolate SN caused necrotic spots in inoculated leaves without systemic movement, while sub-group I isolate SL infected them systemically without necrosis. Another sub-group I and two other sub-group II isolates behaved like SL in P26849 and P26853 but infected only inoculated leaves of P26858. This suggests that two strain specific hypersensitive resistance specificities are operating against CMV in L. hispanicus. When plants of L. luteus genotypes that gave hypersensitive reactions on graft-inoculation were inoculated with infective sap containing two sub-group I and seven sub-group II isolates, they all responded like L. hispanicus P26858. A strain group concept is proposed for CMV in lupins based on the two hypersensitive specificities found: strain group 1 represented by isolate SN which induces hypersensitivity with both specificities, strain group 2 represented by the three isolates which induced hypersensitivity only with the specificity present in L. luteus and L. hispanicus P26858, strain group 3 by as yet hypothetical isolates that induce hypersensitivity only in presence of the specificity in L. hispanicus P26849 and P26853 that responded just to isolate SN, and strain group 4 by isolate SL which overcomes both specificities. When F₂ progeny plants from crosses between hypersensitive and susceptible L. luteus parents were inoculated with isolate SN, the resistance segregated with a 3:1 ratio (hypersensitive:susceptible), suggesting that a single dominant hypersensitivity gene, Ncm-1, is responsible. As gene Ncm-1 had broad specificity and was not overcome by any of the five CMV isolates from lupins tested, it is valuable for use in breeding CMV resistant L. luteus cultivars.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. II. Linkage relations and utility of a dominant gene for lethal systemic necrosis to soybean mosaic virus

Summary A single dominant factor, Hss, that conditions a rapid lethal necrotic response to soybean mosaic virus (SMV) has been identified in Phaseolus vulgaris L. cv. Black Turtle Soup, line BT-1. Inoculated plants carrying this factor developed pinpoint necrotic lesions on inoculated tissue followed by systemic vascular necrosis and plant death within about 7 days, regardless of ambient temperature. BT-1 also carries dominant resistance to potyviruses attributed to the tightly linked or identical factors, I, Bcm, Cam, and Hsw, so linkage with Hss was evaluated. No recombinants were identified among 381 F₃ families segregating for potyvirus susceptibility, thus if Hss is a distinct factor, it is tightly linked to I, Bcm, Cam, and Hsw. BT-1 was also crossed reciprocally with the line Great Northern 1140 (GN 1140) in which the dominant gene, Smv, for systemic resistance to SMV was first identified. Smv and Hss segregated independently and are co-dominant. The (GN 1140 x BT-1) F₁ populations showed a seasonal shift of the codominant phenotype. Evaluation of the (GN 1140 x BT-1) F₂ population under conditions where Smv is partially dominant allowed additional phenotypic classes to be distinguished. Pathotype specificity has not been demonstrated for either Smv or Hss. Genotypes that are homozygous for both dominant alleles are systemically resistant to the virus and in addition show undetectable local viral replication or and no seed transmission. This work demonstrates that a gene which conditions a systemic lethal response to a pathogen may be combined with additional gene(s) to create an improved resistant phenotype.     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

Antisense RNA approach targeting Rep gene of Mungbean yellow mosaic India virus to develop resistance in soybean

The Yellow mosaic disease is caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus of the family Geminiviridae. Yellow mosaic disease (YMD) is a major constraint to the production of soybean in South-East Asia. In India, yield losses of 10–88% had been reported due to YMD of soybean. An effort has been made to generate resistant soybean plants, by a construct targeting replication initiation protein (Rep) gene sequences of MYMIV. A construct containing the sequences of Rep gene (566?bp) in antisense orientation was used to transform cotyledonary node explants of three soybean cultivars (JS 335, JS 95-60 and NRC 37). Transformation efficiencies of 0.2, 0.21 and 0.24% were obtained with three soybean cultivars, JS 335, JS 95-60 and NRC 37, respectively. The presence of transgene in T₁ plants was confirmed by polymerase chain reaction (PCR) and sequence analysis. The level of resistance was observed by challenge inoculation with the virus in T₁ lines. The inheritance of transgene showed classical Mendelian pattern in six transgenic lines.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. IV. Inheritance, linkage relations, and environmental effects on systemic resistance to four potyviruses

We have examined the genetics of systemic resistance in Phaseolus vulgaris to azuki bean mosaic virus (AzMV) and cowpea aphid-borne mosaic virus (CABMV) and the relationship of this resistance to a phenotypically similar resistance to watermelon mosaic virus (WMV) and soybean mosaic virus (SMV). In P. vulgaris cv Great Northern 1140 (GN1140), resistance to SMV and WMV has been attributed to the genes Smv and Wmv, respectively, which have been shown to segregate as a unit. Systemic resistance to AzMV is conferred by two incompletely dominant alleles, Azm1 and Azm2, at unlinked loci. At least three resistance alleles must be present at these two loci for systemic resistance to be expressed in the plant. Systemic resistance to CABMV in GN 1140 is conditioned by a dominant allele that has been designated Cam2. Under some environmental conditions, a recessive allele at an unlinked locus, cam3, also controls a resistant response to CABMV. Resistance to AzMV and CABMV does not assort independently from Wmv/Smv, but also does not consistently cosegregate, suggesting that perhaps in each case one of the factors involved in resistance is associated with Smv/Wmv.     

Coat protein-mediated resistance to Turnip mosaic virus in oilseed rape (Brassica napus)

Oilseed rape (Brassica napus) lines transformedwith the coat protein (CP) gene of Turnip mosaic virus(TuMV) were used to determine the effectiveness of resistance to TuMV mediatedby CP RNA or coat protein. Lines with one, two, or more copies of transgeneswere produced. T₂ and T₃ lines containing the CP genewitha functional start codon synthesised coat protein and showed high, but variablelevels of resistance to TuMV (21–96% resistant plants per line). TheT₁ and T₂ progeny of all lines carrying the CP gene withamutated start codon so that RNA but not protein was expressed, were assusceptible to TuMV as controls. Thus, in these experiments we were able toinduce CP-mediated resistance, but not RNA-mediated resistance.     

Oligogenic inheritance for resistance to Zucchini yellow mosaic virus in Cucurbita pepo

‘True French’ is an open‐pollinated cultivar of the Zucchini (Courgette) Group of Cucurbita pepo and is susceptible to Zucchini yellow mosaic virus (ZYMV). Using C. moschata‘Menina’ as the source of ZYMV resistance and following six generations of backcrossing, a true‐breeding line nearly isogenic to ‘True French’, designated 381e, was recovered that carried ZYMV resistance, albeit not at as high a level as in ‘Menina’. ‘True French’ and accession 381e were crossed, and their reciprocal F₁, F², and backcross progenies were grown in a chamber and inoculated with a highly virulent, non‐aphid‐transmissible strain of ZYMV. Nearly all F¹ plants and all plants of the backcross to 381e were classified as resistant. Segregation to resistant and susceptible individuals occurred in the backcross to the susceptible parent, in accordance with a 3:5 three‐gene ratio of resistant: susceptible. The F₂ segregated in accordance with a ratio of 45 resistant : 19 susceptible, which would be obtained if there was one major gene for resistance, Zym‐1 (Zym), and two other genes, herein designated Zym‐2 and Zym‐3, both of which for complementary to Zym‐1. The presence of Zym‐1 and either Zym‐2 or Zym‐3 is necessary for resistance to be expressed in young plants, but the presence of all three might be necessary for resistance to continue to be expressed during subsequent development of the plants. Evidently, Zym‐2 and Zym‐3 are ubiquitous in C. moschata but their susceptible alleles are much more common in C. pepo. As the level of resistance of 381e to ZYMV is not as high as that of C. moschata‘Menina’, additional, as yet unidentified, genes must be involved in conferring high resistance to this virus.     

Resistance of transgenic tomato to cucumber mosaic cucumovirus under field conditions

Since the summer of 1993, transgenic tomato plants expressing the coat protein (CP) genes of cucumber mosaic cucumovirus have been tested under field conditions to assess the level of resistance and agronomic performance. Trials were performed in different areas in Italy and the target virus in the majority of tests was spread naturally by the indigenous aphid populations. Twenty-three homozygous lines of variety UC82B, transformed to contain four different CP genes of CMV, were evaluated. The lines were preselected for CP expression, single gene copy, and virus resistance in growth chamber experiments. In general, CMV resistance was confirmed under field conditions though resistance in the field was less effective than what was observed in growth chamber experiments. The resistance observed in multi-year and multi-location experiments is of commercial value for several of the most resistant lines. Engineered resistance upon transfer to Italian varieties by breeding or direct transformation will be used in tomato production in Italy or elsewhere. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on resistance of pea to pea seed borne mosaic virus and new pathotypes

Studies were carried out to search for virulent pathotypes of pea seed borne mosaic virus (PSbMV) on pea and to explore new sources of resistance in French and Indian pea collections. A virulent pathotype, PSbMV-Pi, capable of partially overcoming the recessively resistant gene sbm 1, was identified for the first time in an Indian pea line. PSbMV-Pi did not produce visible symptoms on sbm 1 lines, on which it had a reduced multiplication and could no longer be detected by ELISA seven wk after inoculation. However, it multiplied normally on the susceptible cultivars and could be differentiated from other strains on a set of strain differentials. Also, another strain, PSbMV-Pv, of the virulent pathotype, that appeared to multiply slightly better than PSbMV-Pi on sbm 1 lines, was recovered from the local strain of PSbMV.
Five new sources of resistance to PSbMV were screened from the Indian pea collection by a rapid screening based on the assumption that, in germplasm collections, the virus is generalised to all susceptible lines by aphid vectors. The initial step of testing a few testas of each germplasm line in ELISA and the rejection of positive lines eliminated 85% of the germplasm before further testing in the field. The inheritance studies on four of the five resistant lines lead to reidentification of the sbm 1 gene. The sbm 1 lines behaved fairly well under heavy inoculum pressure in the pea fields in 1989.     

Development of PCR markers linked to resistance to wheat streak mosaic virus in wheat

Wheat streak mosaic virus (WSMV), vectored by the wheat curl mite (Acer tulipae), is an important disease of wheat (Triticum aestivum L.) in the North American Great Plains. Resistant varieties have not been developed for two primary reasons. First, useful sources of resistance have not been available, and second, field screening for virus resistance is laborious and beyond the scope of most breeding programs. The first problem may have been overcome by the development of resistance to both the mite and the virus by the introgression of resistance genes from wild relatives of wheat. To help address the second problem, we have developed polymerase chain reaction (PCR) markers linked to the WSMV resistance gene Wsm1. Wsm1 is contained on a translocated segment from Agropyron intermedium. One sequence-tagged-site (STS) primer set (WG232) and one RAPD marker were found to be linked to the translocation containing Wsm1. The diagnostic RAPD band was cloned and sequenced to allow the design of specific PCR primers. The PCR primers should be useful for transferring Wsm1 into locally adapted cultivars.This is Journal Series No. J-4041 of the Montana Agricultural Experiment Station