Response of maize (Zea mays L.) lines carrying Wsm1, Wsm2, and Wsm3 to the potyviruses Johnsongrass mosaic virus and Sorghum mosaic virus

Maize dwarf mosaic disease is one of the most important viral diseases of maize (Zea mays L.) throughout the world. It is caused by several virus species in the family Potyviridae, genus Potyvirus, including Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), Johnsongrass mosaic virus (JGMV) and Sorghum mosaic virus (SrMV). Resistance to another member of the family Potyviridae, Wheat streak mosaic virus (WSMV), is conferred by three alleles (Wsm1, Wsm2, Wsm3) in the maize inbred line Pa405, and these or closely linked genes were previously shown to confer resistance to the potyviruses MDMV and SCMV. In this study, we assessed whether Wsm alleles are linked to resistance to JGMV and SrMV. Near isogenic lines (NILs) carrying one or two of the Wsm alleles introgressed into the susceptible line Oh28 and F1 progeny from NIL × Oh28 were tested for their response to JGMV and SrMV. Our results indicate that Wsm1 provides resistance to both JGMV and SrMV in a dose-dependent manner. Wsm2 and Wsm3 each provide limited resistance, and combining Wsm alleles enhances that resistance.     

Responses of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) near isogenic lines carrying <Emphasis Type="Italic">Wsm1</Emphasis>, <Emphasis Type="Italic">Wsm2,</Emphasis> and <Emphasis Type="Italic">Wsm3</Emphasis> to three viruses in the Potyviridae

Genes on chromosomes six (Wsm1), three (Wsm2) and ten (Wsm3) in the maize (Zea mays L.) inbred line Pa405 control resistance to Wheat streak mosaic virus (WSMV), and the same or closely linked genes control resistance to Maize dwarf mosaic virus (MDMV) and Sugarcane mosaic virus (SCMV). Near isogenic lines (NIL) carrying one or two of the genes were developed by introgressing regions of the respective chromosomes into the susceptible line Oh28 and tested for their responses to WSMV, MDMV, and SCMV in the field and greenhouse. F₁ progeny from NIL × Oh28 were also tested. Wsm1, or closely linked genes, provided resistance to all three viruses, as determined by symptom incidence and severity. Wsm2 and Wsm3 provided resistance to WSMV. Wsm2 and/or Wsm3 provided no resistance to MDMV, but significantly increased resistance in plants with one Wsm1 allele. NIL carrying Wsm1, Wsm2, or Wsm3 had similar SCMV resistance in the field, but NIL with Wsm2 and Wsm3 were not resistant in the greenhouse. Addition of Wsm2 to Wsm1 increased SCMV resistance in the field. For all viruses, symptom incidence was higher in the greenhouse than in the field, and relative disease severity was higher in the greenhouse for WSMV and MDMV. An Italian MDMV isolate and the Ohio SCMV infected the Wsm1 NIL, while the Ohio MDMV and Seehausen SCMV isolates did not. Our results indicate that the three genes, or closely linked loci, provide virus resistance. Resistance conferred by the three genes is influenced by interactions among the genes, the virus species, the virus isolate, and the environment.     

Identification of quantitative trait loci controlling resistance to maize chlorotic dwarf virus

Ineffective screening methods and low levels of disease resistance have hampered genetic analysis of maize (Zea mays L.) resistance to disease caused by maize chlorotic dwarf virus (MCDV). Progeny from a cross between the highly resistant maize inbred line Oh1VI and the susceptible inbred line Va35 were evaluated for MCDV symptoms after multiple virus inoculations, using the viral vector Graminella nigrifrons. Symptom severity scores from three rating dates were used to calculate area under the disease progress curve (AUDPC) scores for vein banding, leaf twist and tear, and whorl chlorosis. AUDPC scores for the F₂ population indicated that MCDV resistance was quantitatively inherited. Genotypic and phenotypic analyses of 314 F₂ individuals were compared using composite interval mapping (CIM) and analysis of variance. CIM identified two major quantitative trait loci (QTL) on chromosomes 3 and 10 and two minor QTL on chromosomes 4 and 6. Resistance was additive, with alleles from Oh1VI at the loci on chromosomes 3 and 10 contributing equally to resistance.     

Impact of 9 years of Bt-maize cultivation on the distribution of maize viruses

This study assesses the effect of Bt-maize on the distribution of maize viruses. Random surveys were conducted in Spain between 2001 and 2006 to evaluate the occurrence of maize viruses in Bt-maize cultivation areas and in areas where this crop had not been introduced. Maize dwarf mosaic virus (MDMV) was the predominant virus in Bt-areas, and Maize rough dwarf virus (MRDV) was the most predominant one in non-Bt-areas, with MRDV an emergent virus in both types of areas. A decline in the occurrence of MDMV and an increase in that of Sugarcane mosaic virus was observed in Bt-areas. Additionally, data obtained over 6 years in experimental fields showed non-significant differences between the infection rates exhibited by two generations of Bt varieties and the non-transformed isogenics varieties for any of the viruses. Our data suggest that differences in virus distribution are linked to the genetic background of the maize varieties and the distribution of virus reservoirs rather than to Bt-maize cultivation.     

RNA interference-mediated resistance to maize dwarf mosaic virus

Maize dwarf mosaic virus (MDMV) is a widespread pathogen that causes serious yield loss to maize crops. A hairpin RNA expression vector was constructed herein to overcome the low efficiency of cultural protection against MDMV and to improve the MDMV resistance mediated by a shorter transgenic inverted-repeat sequence. This expression vector contained a 451 bp inverted-repeat sequence, homologous to the protease gene (P1) of MDMV. It was used for the Agrobacterium tumefaciens-mediated transformation of maize calli induced from a susceptible inbred line. A total of 17 T₂ transgenic lines were identified by both specific PCR amplification and Southern blot hybridization. Of these lines, 15 were evaluated for MDMV resistance in inoculation field trials under two environments. The relative replication levels of the P1 gene were analyzed by quantitative real-time (qRT)-PCR. Results demonstrated that all of the 15 T₂ lines showed an enhanced resistance to MDMV in comparison with that of the non-transformed parent line. Six lines were deemed to be ‘resistant’ with an average disease index below 25 %, which was not significantly different from that of the resistant control. The relative replication levels of the virus gene were significantly reduced in these resistant T₂ transgenic lines. The efficiency of virus gene silencing was directly related to the transgene copy numbers presented in these transgenic lines.     

Genetic analysis of resistance in Brussels sprout to cauliflower mosaic and turnip mosaic viruses

A total of 28 inbred lines of Brussels sprout were assessed in the glasshouse for their reaction to inoculation with cauliflower mosaic (CaMV) or turnip mosaic (TuMV) virus. There was significant variation for resistance to both viruses. From the 28 inbred lines parents were chosen for two 9 × 9 diallel crossing programmes. The parents and their F₁ progeny were assessed for their reaction to CaMV or TuMV in the field. There was significant additive and non-additive (dominance) variation but no maternal effects. Resistance to both viruses was generally dominant but with some evidence of a recessive gene for resistance to CaMV. Resistance to TuMV and CaMV was apparently controlled by at least four genes and two genes respectively. The heritability of resistance to each virus was high. The implications for breeding F₁ hybrid Brussels sprout cultivars are discussed.     

Quantitative trait loci for resistance to Maize rayado fino virus

Maize rayado fino virus (MRFV) causes one of the most important virus diseases of maize in regions of Mexico, Central and South America, where it causes moderate to severe yield losses. The virus is found from the southern USA to northern Argentina where its vector, the maize leafhopper Dalbulus maidis, is present. Although resistance to MRFV has been identified in tropical maize lines, little was known about genes or quantitative trait locus (QTL) conferring resistance to MRFV. In order to identify the location of genes conferring resistance to MRFV, two recombinant inbred line mapping populations that segregated for MRFV resistance were inoculated using viruliferous leafhoppers, and their responses to virus inoculation were evaluated under greenhouse conditions 7, 14 and 21 days post inoculation. A QTL explaining up to 23 % of the total phenotypic variance was mapped on chromosome 10 in both populations, with similar genetic and physical positions identified in the two populations. The magnitude of the QTL effect and the validation in two independent populations suggests that resistance to MFRV could be transferred into elite breeding lines to develop resistant cultivars.     

Epistatic determinism of durum wheat resistance to the wheat spindle streak mosaic virus

Key message

The resistance of durum wheat to the Wheat spindle streak mosaic virus (WSSMV) is controlled by two main QTLs on chromosomes 7A and 7B, with a huge epistatic effect.

Abstract

Wheat spindle streak mosaic virus (WSSMV) is a major disease of durum wheat in Europe and North America. Breeding WSSMV-resistant cultivars is currently the only way to control the virus since no treatment is available. This paper reports studies of the inheritance of WSSMV resistance using two related durum wheat populations obtained by crossing two elite cultivars with a WSSMV-resistant emmer cultivar. In 2012 and 2015, 354 recombinant inbred lines (RIL) were phenotyped using visual notations, ELISA and qPCR and genotyped using locus targeted capture and sequencing. This allowed us to build a consensus genetic map of 8568 markers and identify three chromosomal regions involved in WSSMV resistance. Two major regions (located on chromosomes 7A and 7B) jointly explain, on the basis of epistatic interactions, up to 43% of the phenotypic variation. Flanking sequences of our genetic markers are provided to facilitate future marker-assisted selection of WSSMV-resistant cultivars.

     

Linkage between Frl (Fusarium oxysporum f.sp. radicis-lycopersici resistance) and Tm-2 (tobacco mosaic virus resistance-2) loci in tomato (Lycopersicon esculentum)

A study was carried out on the linkage relationship between the Frl locus carrying resistance to Fusarium oxysporum f.sp. radicis-lycopersici and the Tm-2 locus carrying resistance to several races of tobacco mosaic virus in the tomato inbred line IRB-301-31. The inbred line Motelle (Frl⁺/Frl⁺, Tm-2⁺/Tm-2⁺) was crossed with the inbred line IRB-301-31 (Frl/Frl, Tm-2/Tm-l). The resulting 222 F₂ plants were selfed, and from each F₃ family groups of 15–60 seedlings were tested for resistance to either F. oxysporum f.sp. radicis-lycopersici or tobacco mosaic virus race 0. Segregation data indicated a very tight linkage between Frl and Tm-2, equal to 5.1 ± 1.07 map units.     

两个玉米矮花叶病显性互补抗病基因的发现和定位

玉米矮花叶病是世界普通发生危害严重的玉米病毒病害之一,迄今为止,只有少数几个抗病基因被发现并定位,优良自交系四一是鉴定出定的玉米筹花叶病新抗源,它表现为全生育抗性,通过连续两年的经典遗传学研究发现,四一的成株期抗性表现为一种新的抗病遗传模式,该抗性是由两个显性互补抗病基因控制,87对微卫星标记分析进一步证实了以上推论,并把两个抗病基因分别定位在第三和第六染色体上,第三染色体上的抗病基因与微卫星标记phi029相距14.5cM,第六染色体上的抗病基因与微卫星标记phil26相距7.2cM.     

Identification of maize lethal necrosis disease causal viruses in maize and suspected alternative hosts through small RNA profiling

Maize lethal necrosis disease (MLND) is a devastating viral disease of maize caused by double infection with Maize chlorotic mottle virus (MCMV) and any one of the Potyviridae family members. Management of MLND requires effective resistance screening and surveillance tools. In this study, we report the use of small RNA (sRNA) profiling to detect MLND causal viruses and further the development of alternative detection markers for use in routine surveillance of the disease-causing viruses. Small RNAs (sRNAs) originating from five viruses namely MCMV, Sugarcane mosaic virus (SCMV), Maize streak virus (MSV), Maize-associated totivirus (MATV) and Maize yellow mosaic virus (MYMV) were assembled from infected maize samples collected from MLND hot spots in Kenya. The expression of the identified viral domains was further validated using quantitative real-time PCR. New markers for the detection of some of the MLND causal viruses were also developed from the highly expressed domains and used to detect the MLND-causative viruses in maize and alternative hosts. These findings further demonstrate the potential of using sRNAs especially from highly expressed viral motifs in the detection of MLND causal viruses. We report the validation of new sets of primers for use in detection of the most common MLND causal viruses MCMV and SCMV in East Africa.     

Expression of an extracellular ribonuclease gene increases resistance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

     

Genome-wide association analysis identified SNPs closely linked to a gene resistant to Soil-borne wheat mosaic virus

Key message

Using association and linkage mapping, two SNP markers closely linked to the SBWMV resistance gene on chromosome 5D were identified and can be used to select the gene in breeding.

Abstract

Soil-borne wheat mosaic virus (SBWMV) disease is a serious viral disease of winter wheat growing areas worldwide. SBWMV infection can significantly reduce grain yield up to 80 %. Developing resistant wheat cultivars is the only feasible strategy to reduce the losses. In this study, wheat Infinium iSelect Beadchips with 9 K wheat SNPs were used to genotype an association mapping population of 205 wheat accessions. Six new SNPs from two genes were identified to be significantly associated with the gene for SBWMV resistance on chromosome 5D. The SNPs and Xgwm469, an SSR marker that has been reported to be associated with the gene, were mapped close to the gene using F₆-derived recombinant inbred lines from the cross between a resistant parent ‘Heyne’ and a susceptible parent ‘Trego’. Two representative SNPs, wsnp_CAP11_c209_198467 and wsnp_JD_c4438_5568170, from the two linked genes in wheat were converted into KBioscience Competitive Allele-Specific Polymerase assays and can be easily used in marker-assisted selection to improve wheat resistance to SBWMV in breeding.     

Simple sequence repeat markers linked to QTL for resistance to Watermelon mosaic virus in melon

A population of recombinant inbred lines (RIL) derived from a cross between the Watermelon mosaic virus (WMV) resistant genotype TGR-1551 and the susceptible Spanish cultivar ‘Bola de Oro’ has been evaluated for WMV resistance in spring, fall and growth chamber conditions. The quantitative trait loci (QTL) analyses detected one major QTL (wmv) on linkage group (LG) XI close to the microsatellite marker CMN04_35. This QTL controls the resistance to WMV in the three environmental conditions evaluated. Other minor QTLs affecting the severity of viral symptoms were identified, but they were not detected in all the assayed environments. The screening of the marker CMN04_35 in an F₂ progeny, derived from the same cross, confirmed the effect of this QTL on the expression of WMV resistance also in early generations, which evidences the usefulness of this marker for a marker assisted selection program.     

High-resolution mapping of zym, a recessive gene for Zucchini yellow mosaic virus resistance in cucumber

Key message

Using a high-resolution mapping approach, we identified a candidate gene for ZYMV resistance in cucumber. Our findings should assist the development of high-versatility molecular markers for MAS for ZYMV resistance.

Abstract

Zucchini yellow mosaic virus (ZYMV) causes significant disease, which leads to fruit yield loss in cucurbit crops. Since ZYMV resistance is often inherited recessively in cucumber, marker-assisted selection (MAS) is a useful tool for the development of resistant cucumber cultivars. Using 128 families of an F_2:3 population derived from a cross between susceptible ‘CS-PMR1’ and resistant ‘A192-18’ cucumber inbred lines, we confirmed that ZYMV resistance is conferred by a single recessive locus: zym ^A192-18 . We constructed a cucumber genetic linkage map that included 125 simple sequence repeat (SSR) markers segregating into 7 linkage groups (chromosomes). The zym ^A192-18 locus was mapped to chromosome 6, at genetic distances of 0.9 and 1.3 cM from two closely linked SSR markers. For high-resolution genetic mapping, we identified new molecular markers cosegregating with the zym ^A192-18 locus; using cucumber genomic and molecular marker resources and screening an F₂ population of 2,429 plants, we narrowed down the zym ^A192-18 locus to a <50-kb genomic region flanked by two SSR markers, which included six candidate genes. Sequence analysis of the candidate genes’ coding regions revealed that the vacuolar protein sorting-associated protein 4-like (VPS4-like) gene had two SNPs between the parental lines. Based on SNPs of the VPS-4-like gene, we developed zym ^A192-18 -linked DNA markers and found that genotypes associated with these markers were correlated with the ZYMV resistance phenotype in 48 cucumber inbred lines. According to our data, the gene encoding VPS4-like protein is a candidate for the zym ^A192-18 locus. These results may be valuable for MAS for ZYMV resistance in cucumber.     

Identification and genetic mapping for <Emphasis Type="Italic">rht-DM</Emphasis>, a dominant dwarfing gene in mutant semi-dwarf maize using QTL-seq approach

Semi-dwarfism is an agronomically important trait in breeding for stable high yields and for resistance to damage by wind and rain (lodging resistance). Many QTLs and genes causing dwarf phenotype have been found in maize. However, because of the yield loss associated with these QTLs and genes, they have been difficult to use in breeding for dwarf stature in maize. Therefore, it is important to find the new dwarfing genes or materials without undesirable characters. The objectives of this study were: (1) to figure out the inheritance of semi-dwarfism in mutants; (2) mapping dwarfing gene or QTL. Maize inbred lines ‘18599’ and ‘DM173’, which is the dwarf mutant derived from the maize inbred line ‘173’ through ⁶⁰Co-γ ray irradiation. F₂ and BC₁F₁ population were used for genetic analysis. Whole genome resequencing-based technology (QTL-seq) were performed to map dwarfing gene and figured out the SNP markers in predicted region using dwarf bulk and tall bulk from F₂ population. Based on the polymorphic SNP markers from QTL-seq, we were fine-mapping the dwarfing gene using F₂ population. In F₂ population, 398 were dwarf plants and 135 were tall plants. Results of χ² tests indicated that the ratio of dwarf plants to tall plants was fitted to 3:1 ratio. Furthermore, the χ² tests of BC₁F₁ population showed that the ratio was fitted to 1:1 ratio. Based on QTL-seq, the dwarfing gene was located at the region from 111.07 to 124.56 Mb of chromosome 9, and we named it rht-DM. Using traditional QTL mapping with SNP markers, the rht-DM was narrowed down to 400 kb region between SNP-21 and SNP-24. The two SNPs were located at 0.43 and 0.11 cM. Segregation analysis of F₂ and BC₁F₁ indicated that the dwarfing gene was likely a dominant gene. This dwarfing gene was located in the region between 115.02 and 115.42 Mb on chromosome 9.     

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world''s most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials ⁹, vaccines ^{14, 15}, electronic materials, chemical tools, and molecular electronic containers^{4, 5, 10, 11, 16, 18, 12}.In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups ⁸. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications ⁸. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed ¹³. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus ³, Carnation mottle virus ¹², Cowpea chlorotic mottle virus ⁶, Tobacco mosaic virus ¹⁷, Turnip yellow mosaic virus ¹, and MRFV ¹³.     

Inheritance of resistance to watermelon mosaic virus in the cucumber line TMG-1: tissue-specific expression and relationship to zucchini yellow mosaic virus resistance

The inbred cucumber (Cucumis sativus L.) line TMG-1 is resistant to three potyviruses:zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). The genetics of resistance to WMV and the relationship of WMV resistance to ZYMV resistance were examined. TMG-1 was crossed with WI-2757, a susceptible inbred line. F₁, F₂ and backcross progeny populations were screened for resistance to WMV and/or ZYMV. Two independently assorting factors conferred resistance to WMV. One resistance was conferred by a single recessive gene from TMG-1 (wmv-2). The second resistance was conferred by an epistatic interaction between a second recessive gene from TMG-1 (wmv-3) and either a dominant gene from WI-2757 (Wmv-4) or a third recessive gene from TMG-1 (wmv-4) located 20–30 cM from wmv-3. The two resistances exhibited tissue-specific expression. Resistance conferred by wmv-2 was expressed in the cotyledons and throughout the plant. Resistance conferred by wmv-3 + Wmv-4 (or wmv-4) was expressed only in true leaves. The gene conferring resistance to ZYMV appeared to be the same as, or tightly linked to one of the WMV resistance genes, wmv-3.     

Identification of markers tightly linked to<Emphasis Type="Italic"> sbm</Emphasis> recessive genes for resistance to<Emphasis Type="Italic"> Pea seed-borne mosaic virus</Emphasis>

Two virus resistance loci on linkage groups II and VI have provided the only sources of natural resistance against Pea seed-borne mosaic virus (PSbMV, Potyviridae) in the important crop plant Pisum sativum L. A combination of parallel approaches was used to collate linked markers, particularly for sbm-1 resistance on linkage group VI. We have identified sequences derived from the genes for the eukaryotic translation initiation factors eIF4E and eIF(iso)4E as being very tightly linked to the resistance gene clusters on linkage groups VI and II, respectively. In particular, no recombinants between sbm-1 and eIF4E were found amongst 500 individuals of an F₂ cross between the BC₄ resistant line (JI1405) and its recurrent susceptible parent Scout. In a different mapping population, the gene eIF(iso)4E was also shown to be linked to sbm-2 on linkage group II. A parallel cDNA-AFLP comparison of pairs of resistant and susceptible lines also identified an expressed tag marker just 0.7 cM from sbm-1. eIF4E and eIF(iso)4E have been associated with resistance to related viruses in other hosts. This correlation strengthens the use of our markers as valuable tools to assist in breeding multiple virus resistances into peas, and identifies potential targets for resistance gene identification in pea.Communicated by C. Möllers