Caracterização dos mecanismos de defesa do acesso PI414723 de Cucumis melo L. ao Zucchini yellow mosaic virus e seleção de candidatos a genes de resistência no locus Zym-1

Characterization of defense mechanisms of PI414723 accession from Cucumis melo L. to the Zucchini yellow mosaic virus and selection of resistance candidate genes at the Zym-1 locus The zucchini yellow mosaic virus (ZYMV) is a pathogen that is able to severely reduce the yield of melon plants. The use of genetic control is the most efficient way of management for this virus, and the PI414723 (PI) melon accession is the only source of resistance effectively used in breeding programs. The PI resistance is controlled by at least a dominant locus called Zym-1. The objective of this study was: i: to characterize the mechanism of PI414723 resistance to ZYMV related to infection, multiplication and/or systemic viral spread and ii: to select resistance candidate genes at the Zym-1 locus using the information from genetic maps associated with the genome sequence of Cucumis melo, and then quantify the expression levels of the selected genes. Therefore, a methodology for absolute quantification of ZYMV by quantitative real-time PCR (RT-qPCR) was developed to compare the number of viral particles between the resistant PI and susceptible 'Védrantais' (Ved) genotypes. To characterize the resistance in PI, the virus was inoculated into one half of the cotyledons of both genotypes, which were divided into inoculated (I) and noninoculated (NI) regions. The virus was quantified in these regions and biological recovery of the viral particles was carried out at 3, 7 and 10 days after inoculation (dai) and in apical leaves at 10 dai. Furthermore, it was evaluated whether the resistance mechanism involves a hypersensitive reaction (HR) at the inoculation site as well as activation of the systemic acquired resistance mechanism (SAR), through the analysis of the expression of genes encoding PR proteins. In all evaluation times and analyzed tissues, viral titers were higher in Ved compared to PI. The virus was not detected in PI apical leaves, but it was highly detected in Ved at 10 dai. HR was observed in PI cotyledons at 12 and 24 hours after inoculation, the same was not observed in Ved cotyledons. The PR1 and PR4 genes were up regulated only in PI after inoculation. Taken together, the results show that the virus infects and multiplies in both genotypes but does not invade PI systemically. In addition, the SAR mechanism is activated in the resistant genotype and the virus is restricted to the inoculation site due to HR. Regarding the selection of candidate genes at the Zym-1 locus, 13 genes were selected on chromosome 2 based on their protein domains involved in virus resistance. The expression of the candidate genes was analyzed in cotyledons at 12 and 24 hours after inoculation (hai) and the virus was quantified at these same periods, as well as at 7 days after inoculation (dai). The viral titer did not vary between genotypes at 12 hai, but was higher in Ved at 24 hai. Two genes were up regulated at 12 hai in PI, which are copies of the RTM2 gene (restricted TEV movement 2), responsible for restricting the virus long distance movement. At 24 hai, two resistance gene analogs (RGA) were down regulated in Ved. One of these RGAs encodes a CC-NB-LRR protein with additional virus recognition domain (Rx-like) and has been up regulated in PI. Therefore, distinct mechanisms are activated in PI414723 in response to ZYMV infection, enabling this PI to resist systemic viral spread.