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.     

Inheritance of resistance to zucchini yellow mosaic virus and watermelon mosaic virus in watermelon

High resistance to zucchini yellow mosaic virus-China strain (ZYMV-CH) and moderate resistance to watermelon mosaic virus (WMV) were found in a selection of PI 595203 (Citrullus lanatus var. lanatus), an Egusi type originally collected in Nigeria. Mixed inoculations showed primarily that these two viruses have no cross-protection. This fact may explain the high frequency of mixed infection often observed in commercial fields. When plants were inoculated with a mixture of the two viruses, the frequency of plants resistant to ZYMV was lower than expected, indicating that WMV infection may reduce the ability of a plant to resist ZYMV. We studied inheritance of resistance to ZYMV-CH and WMV, using crosses between a single-plant selection of PI 595203 and the ZYMV-susceptible watermelon inbreds 9811 and 98R. According to virus ratings of the susceptible parents, the resistant parent, and the F1, F2, and BC1 generations, resistance to ZYMV-CH was conferred by a single recessive gene, for which the symbol zym-CH is suggested. The high tolerance to WMV was controlled by at least two recessive genes.     

Genetic linkage map of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) and localization of a major QTL for powdery mildew resistance

Powdery mildew caused by Podosphaera xanthii has become a major problem in melon since it occurs all year round irrespective of the growing system. The TGR-1551 melon genotype was found to be resistant to several melon diseases, among them powdery mildew. However, the corresponding resistance genes have been never mapped. We constructed an integrated genetic linkage map using an F2 population derived from a cross between the multi-resistant genotype TGR-1551 and the susceptible Spanish cultivar ‘Bola de Oro’. The map spans 1,284.9 cM, with an average distance of 3.6 cM among markers, and consists of 354 loci (188 AFLP, 39 RAPD, 111 SSR, 14 SCAR/CAPS/dCAPS, and two phenotypic traits) distributed in 14 linkage groups. QTL analysis identified one major QTL (Pm-R) on LG V for resistance to races 1, 2, and 5 of powdery mildew. The PM4-CAPS marker is closely linked to the Pm-R QTL at a genetic distance of 1.9 cM, and the PM3-CAPS marker is located within the support interval of this QTL. These codominant markers, together with the map information reported here, could be used for melon breeding, and particularly for genotyping selection of resistance to powdery mildew in this vegetable crop species.     

Ribozyme genes protecting transgenic melon plants against potyviruses

Potyviruses are the most important viral pathogens of crops worldwide. Under a contract with Gene Shears Pty Limited, we are using ribozyme genes to protect melon plants against two potyviruses: WMV2 and ZYMV. Different polyribozyme genes were designed, built and introduced into melons plants. Transgenic melon plants containing a resistance gene were obtained and their progeny was challenged by the appropriate virus. Most of the genes tested conferred some degree of resistance to the viruses in glasshouse trials. Melon plants from one family containing one gene directed against WMV2 were also field-trialed on small plots under natural infection pressure and were found immune to WMV2. Field trial is in progress for plants containing genes against ZYMV. Some of the ribozyme genes used in the plants were also assayed in a transient expression system in tobacco cells. This enabled us to study the sequence discrimination capacity of the ribozyme in the case of one ribozyme target site. We found that a mutated target GUG (non cleavable) was less susceptible to inhibition by the ribozyme gene than the corresponding wild type target GUA (cleavable). Work is now in progress to incorporate multiple resistance genes in melon plants, in constructs designed in compliance with the evolving European regulations concerning transgenic plants. The use of ribozyme genes to protect plants against viruses provides an alternative to the technologies currently used for protecting crops against viruses, based on the concept of Pathogen Derived Resistance (see for example 14). In the light of concerns expressed by some plant virologists (13) about the use of viral genes in transgenic plants, it may be that ribozyme genes will find many uses in this area of agricultural biotechnology.     

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.     

Inheritance and QTL mapping of glandular trichomes type I density in <Emphasis Type="Italic">Cucumis melo</Emphasis> L.

To determine the genetic basis of the density of glandular trichomes type I in melon, a six-generation family and a recombinant inbred lines (F₇) population have been tested for the character; both genetic groups have been obtained from a cross between the Zimbabwean melon line TGR-1551 (high density of glandular trichomes) and the Spanish cultivar ‘Bola de Oro’ (low density of glandular trichomes). Classic quantitative genetic analysis indicated an oligogenic model for the inheritance of the character with an important additive component and strong epistatic relationships between loci. The quantitative trait locus (QTL) analyses revealed that the character is controlled by one major QTL, tric11, which explained between 23.8 and 58.7 % of its phenotypic variance; other minor QTLs of weaker effect and dependent of the assayed growing conditions were also found to be involved in the character expression. A codominant marker, the microsatellite ECM63, was found to be linked to the major QTL tric11. The validation of this marker in advanced backcrosses confirmed that the TGR-1551 alleles for this QTL increased glandular trichome density. The validation of the detected QTL was also done in other genetic backgrounds, which indicated that ECM63 could be effective in the selection of plants with high density of glandular trichomes type I.     

Transgenic Melon and Squash Expressing Coat Protein Genes of Aphid-borne Viruses do not Assist the Spread of an Aphid Non- transmissible Strain of Cucumber Mosaic Virus in the Field

Transgenic melon and squash containing the coat protein (CP) gene of the aphid transmissible strain WL of cucumber mosaic cucumovirus (CMV) were grown under field conditions to determine if they would assist the spread of the aphid non-transmissible strain C of CMV, possibly through heterologous encapsidation and recombination. Transgenic melon were susceptible to CMV strain C whereas transgenic squash were resistant although the latter occasionally developed chlorotic blotches on lower leaves. Transgenic squash line ZW-20, one of the parents of commercialized cultivar Freedom II, which expresses the CP genes of the aphid transmissible strains FL of zucchini yellow mosaic (ZYMV) and watermelon mosaic virus 2 (WMV 2) potyviruses was also tested. Line ZW-20 is resistant to ZYMV and WMV 2 but is susceptible to CMV. Field experiments conducted over two consecutive years showed that aphid-vectored spread of CMV strain C did not occur from any of the CMV strain C-challenge inoculated transgenic plants to any of the uninoculated CMV-susceptible non- transgenic plants. Although CMV was detected in 3% (22/764) of the uninoculated plants, several assays including ELISA, RT- PCR-RFLP, identification of CP amino acid at position 168, and aphid transmission tests demonstrated that these CMV isolates were distinct from strain C. Instead, they were non-targeted CMV isolates that came from outside the field plots. This is the first report on field experiments designed to determine the potential of transgenic plants expressing CP genes for triggering changes in virus-vector specificity. Our results indicate that transgenic plants expressing CP genes of aphid transmissible strains of CMV, ZYMV, and WMV 2 are unlikely to mediate the spread of aphid non-transmissible strains of CMV. This finding is of practical relevance because transgenic crops expressing the three CP genes are targeted for commercial release, and because CMV is economically important, has a wide host range, and is widespread worldwide.     

Soybean RNA interference lines silenced for eIF4E show broad potyvirus resistance

Soybean mosaic virus (SMV), a potyvirus, is the most prevalent and destructive viral pathogen in soybean-planting regions of China. Moreover, other potyviruses, including bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), also threaten soybean farming. The eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in controlling resistance/susceptibility to potyviruses in plants. In the present study, much higher SMV-induced eIF4E1 expression levels were detected in a susceptible soybean cultivar when compared with a resistant cultivar, suggesting the involvement of eIF4E1 in the response to SMV by the susceptible cultivar. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that soybean eIF4E1 interacted with SMV VPg in the nucleus and with SMV NIa-Pro/NIb in the cytoplasm, revealing the involvement of VPg, NIa-Pro, and NIb in SMV infection and multiplication. Furthermore, transgenic soybeans silenced for eIF4E were produced using an RNA interference approach. Through monitoring for viral symptoms and viral titers, robust and broad-spectrum resistance was confirmed against five SMV strains (SC3/7/15/18 and SMV-R), BCMV, and WMV in the transgenic plants. Our findings represent fresh insights for investigating the mechanism underlying eIF4E-mediated resistance in soybean and also suggest an effective alternative for breeding soybean with broad-spectrum viral resistance.     

西瓜花叶病毒诱导对不同抗性美洲南瓜防卫基因表达的影响

为了明确防卫基因PAL与美洲南瓜抗西瓜花叶病毒(watermelon mosaic virus,WMV)之间的关系,通过室内接种和实时荧光定量PCR技术,测定了WMV侵染对不同抗性美洲南瓜体内防卫基因PAL表达的影响。结果表明:(1)室内测定显示,抗病品种GBRV-8发病率和病情指数(15.6%和14.2)显著低于感病品种‘光板’(91.1%和65.9)。(2)实时荧光定量PCR表明,接种WMV后不同抗感品种不同组织部位PAL基因相对表达量随着接种时间增加,整体呈现出先增加后降低的趋势,而且不同组织部位PAL基因相对表达量总体呈现出叶片较高,叶柄和茎秆次之。(3)接种后5个品种不同组织部位PAL基因相对表达量与对照相比均存在显著差异,且抗病和中抗品种不同组织部位PAL基因相对表达量显著高于感病品种,尤其抗病品种GBRV-8不同组织部位PAL基因相对表达量最高,感病品种光板最低。研究认为,防卫基因PAL表达量与美洲南瓜品种抗病毒病强弱密切相关。     

Blockage of stylet tips as the mechanism of resistance to virus transmission by Aphis gossypii in melon lines bearing the Vat gene

Aphis gossypii is the main virus vector in muskmelon crops. The melon gene Vat confers resistance to non‐persistent virus transmission by this aphid. The mechanism of this resistance is not well understood, but no relationship has been detected between resistance and the probing behaviour of aphids on resistant plants. Results presented here suggest that temporary blockage of aphid stylet tips preventing virus particle release may explain the resistance conferred by Vat gene. We performed experiments in which viruliferous aphids were allowed to probe different sequences of resistant (Vat‐bearing) and/or susceptible melon plants. The results demonstrated that A. gossypii inoculates Cucumber mosaic virus (CMV) efficiently in susceptible plants having previously probed resistant plants, showing that the resistance mechanism is reversible. Furthermore, the infection rate obtained for susceptible plants was the same (25%) regardless of whether the transmitting aphid had come directly from the CMV source or had subsequently probed on resistant plants. This result suggests that virus is not lost from stylet to plant during probing of resistant plants, supporting the temporary blockage hypothesis. We also found that the ability of Myzus persicae to transmit CMV is noticeably reduced after probing on resistant plants, providing evidence that this aphid species also responds to the presence of the Vat gene. Finally, we also found that in probes immediately after virus acquisition M. persicae inoculates resistant plants with CMV more efficiently than susceptible plants, perhaps because the Vat gene product induces increased salivation by this aphid.     

Melon RNA interference (RNAi) lines silenced for Cm-eIF4E show broad virus resistance

Efficient and sustainable control of plant viruses may be achieved using genetically resistant crop varieties, although resistance genes are not always available for each pathogen; in this regard, the identification of new genes that are able to confer broad-spectrum and durable resistance is highly desirable. Recently, the cloning and characterization of recessive resistance genes from different plant species has pointed towards eukaryotic translation initiation factors (eIF) of the 4E family as factors required for the multiplication of many different viruses. Thus, we hypothesized that eIF4E may control the susceptibility of melon (Cucumis melo L.) to a broad range of viruses. To test this hypothesis, Cm-eIF4E knockdown melon plants were generated by the transformation of explants with a construct that was designed to induce the silencing of this gene, and the plants from T2 generations were genetically and phenotypically characterized. In transformed plants, Cm-eIF4E was specifically silenced, as identified by the decreased accumulation of Cm-eIF4E mRNA and the appearance of small interfering RNAs derived from the transgene, whereas the Cm-eIF(iso)4E mRNA levels remained unaffected. We challenged these transgenic melon plants with eight agronomically important melon-infecting viruses, and identified that they were resistant to Cucumber vein yellowing virus (CVYV), Melon necrotic spot virus (MNSV), Moroccan watermelon mosaic virus (MWMV) and Zucchini yellow mosaic virus (ZYMV), indicating that Cm-eIF4E controls melon susceptibility to these four viruses. Therefore, Cm-eIF4E is an efficient target for the identification of new resistance alleles able to confer broad-spectrum virus resistance in melon.     

抗病基因Bdv2抑制大麦黄矮病毒复制和运动的分子证据

小麦-中间偃麦草易位系YW642含有一个源于中间偃麦草7X染色体的抗性基因Bdv2,对大麦黄矮病毒GAV株系具有高度抗性。为有效控制该病毒和阐明抗黄矮病机制,采用半定量RT-PCR的方法,研究了大麦黄矮病毒GAV株系在YW642及其感病姊妹系YW641中积累浓度的差异。分别在接种病毒不同时间、不同部位上取样,用半定量RT-PCR的方法来检测GAV的积累浓度。在接种部位,抗病植株中病毒的浓度远远低于感病植株。在侵染的前5d,抗病植株YW642中病毒会有一定程度的复制和积累,但随后病毒浓度开始下降,接种14—16d时没有检测到病毒;而在感病株系中,病毒积累的浓度远远高于抗病植株,并一直维持一个较高的浓度。在未接种部位．感病植株中可检测到较高浓度的病毒,说明病毒能从接种点很快运动到未接种部位,并大量复制。而在抗病系YW642中,未接种部位始终未检测到病毒。实验结果从分子水平上证明,在抗病植株中BYDV的复制和运动均受到了极大的抑制：这是抗病基因Bdv2与BYDV互作后,激活了一系列防御基因的结果。另外还确定了防御基因诱导表达的时间,为从抗病植株中分离抗病相关基因、研究抗黄矮病机制提供了取样的依据。     

Mutations in the eIF(iso)4G translation initiation factor confer high resistance of rice to Rice yellow mottle virus

We report here evidence of the role that the isoform of the eukaryotic translation initiation factor 4G (eIF(iso)4G) plays in naturally occurring resistance in plant/virus interactions. A genetic and physical mapping approach was developed to isolate the Rymv1 locus controlling the high recessive resistance to Rice yellow mottle virus (RYMV) in the rice (Oryza sativa) variety Gigante. The locus was mapped to a 160-kb interval containing a gene from the eIF(iso)4G family. The stable transformation of a resistant line with the cDNA of this gene, derived from a susceptible variety, resulted in the loss of resistance in transgenic plants. The allelic variability of this gene was analysed in three resistant and 17 susceptible varieties from different cultivated rice species or subspecies. Compared with susceptible varieties, resistant varieties present specific alleles, characterized by either amino acid substitutions or short amino-acid deletions in the middle domain of the protein. The structure of this domain was modelled and showed that the substitutions were clustered on a small surface patch. This suggests that this domain may be involved in an interaction with the virus.     

Mechanism of plant eIF4E-mediated resistance against a Carmovirus (Tombusviridae): cap-independent translation of a viral RNA controlled in cis by an (a)virulence determinant

Translation initiation factors are universal determinants of plant susceptibility to RNA viruses, but the underlying mechanisms are poorly understood. Here, we show that a sequence in the 3' untranslated region (3'-UTR) of a viral genome that is responsible for overcoming plant eIF4E-mediated resistance (virulence determinant) functions as a 3' cap-independent translational enhancer (3'-CITE). The virus/plant pair studied here is Melon necrotic spot virus (MNSV) and melon, for which a recessive resistance controlled by melon eIF4E was previously described. Chimeric viruses between virulent and avirulent isolates enabled us to map the virulence and avirulence determinants to 49 and 26 nucleotides, respectively. The translational efficiency of a luc reporter gene flanked by 5'- and 3'-UTRs from virulent, avirulent and chimeric viruses was analysed in vitro, in wheatgerm extract, and in vivo, in melon protoplasts, showing that: (i) the virulence determinant mediates the efficient cap-independent translation in vitro and in vivo; (ii) the avirulence determinant was able to promote efficient cap-independent translation in vitro, but only when eIF4E from susceptible melon was added in trans, and, coherently, only in protoplasts of susceptible melon, but not in the protoplasts of resistant melon; (iii) these activities required the 5'-UTR of MNSV in cis. Thus, the virulence and avirulence determinants function as 3'-CITEs. The activity of these 3'-CITEs was host specific, suggesting that an inefficient interaction between the viral 3'-CITE of the avirulent isolate and eIF4E of resistant melon impedes the correct formation of the translation initiation complex at the viral RNA ends, thereby leading to resistance.     

Resistance to turnip crinkle virus in Arabidopsis is regulated by two host genes and is salicylic acid dependent but NPR1, ethylene, and jasmonate independent

Inoculation of turnip crinkle virus (TCV) on the resistant Arabidopsis ecotype Dijon (Di-17) results in the development of a hypersensitive response (HR) on the inoculated leaves. To assess the role of the recently cloned HRT gene in conferring resistance, we monitored both HR and resistance (lack of viral spread to systemic tissues) in the progeny of a cross between resistant Di-17 and susceptible Columbia plants. As expected, HR development segregated as a dominant trait that corresponded with the presence of HRT. However, all of the F(1) plants and three-fourths of HR(+) F(2) plants were susceptible to the virus. These results suggest the presence of a second gene, termed RRT, that regulates resistance to TCV. The allele present in Di-17 appears to be recessive to the allele or alleles present in TCV-susceptible ecotypes. We also demonstrate that HR formation and TCV resistance are dependent on salicylic acid but not on ethylene or jasmonic acid. Furthermore, these phenomena are unaffected by mutations in NPR1. Thus, TCV resistance requires a yet undefined salicylic acid-dependent, NPR1-independent signaling pathway.     

Genetics of resistance to cotton leaf curl disease in Gossypium hirsutum L. under field conditions

One hundred and forty two cotton germplasm lines were screened for cotton leaf curl virus symptoms in field evaluations during 2003, 2004, and 2005. Fifty cross combinations involving 30 of these lines classified resistant or susceptible were used for inheritance study of the disease. All the F(1) plants of crosses involving resistant x resistant, resistant x susceptible, and susceptible x resistant parents were resistant, indicating dominant expression of the disease resistance and there were no maternal or cytoplasmic effects detected from reciprocal hybridization. In 22 crosses, 4 types of segregation patterns were obtained in the F(2) generations. A good fit for 15 (resistant):1 (susceptible), 13 (resistant):3 (susceptible), 9 (resistant):7 (susceptible) ratios indicated digenic control of the trait with duplicate dominant, dominant inhibitory, and duplicate recessive epistasis, respectively. Three-gene control with triplicate dominant epistasis was obtained in one of the crosses. This segregation pattern, however, needs further confirmation due to smaller population size. The absence of complementary gene action was obtained in 1 susceptible x susceptible and 27 resistant x resistant crosses as their F(1)s were susceptible and resistant, respectively, and F(2) generation lacked segregation.