RNA-dependent RNA polymerase 1 (RDR1) plays a crucial defense role against plant viruses by secondary amplification of viral double-stranded RNA in the gene-silencing pathway. In this study, it was found that melon (Cucumis melo) encodes four RDR1 genes (CmRDR1a, b, c1 and c2) similar to the CsRDR1 gene family of cucumber (C. sativus). However, in contrast to cucumber, melon harbors a truncated CmRDR1b gene. In healthy plants, CmRDR1a was expressed, whereas the expression of CmRDR1c1/c2 was not detected. CmRDR1a expression level increased 20-fold upon cucumber mosaic virus (CMV) infection and was not increased in melon plants infected with zucchini yellow mosaic virus (ZYMV), cucumber vein yellowing virus (CVYV) and cucumber green mottle mosaic virus (CGMMV). The expression of CmRDR1c1/c2 genes was induced differentially by infection with viruses from different families: high levels of ~340-, 172- and 115-fold increases were induced by CMV, CVYV and CGMMV, respectively, and relatively low-level increases by potyvirus infection (4- to 6-fold). CMV mutants lacking the viral silencing suppressor 2b protein did not cause increased CmRDR1c/c2 expression; knockout of CmRDR1c1/c2 by CRISPR/Cas9 increased susceptibility to CMV but not to ZYMV. Therefore, it is suggested that the sensitivity of melon to viruses from different families is a result of the loss of function of CmRDR1b.
Powdery mildew (PM) diseases may severely limit the production of various crops, including members of to the Cucurbitaceae family. Successful PM infection relies on the Mildew Resistance Locus O (MLO) plant gene family, which encodes susceptibility (S) factors essential for fungus penetration into the host cell. In cucumber, natural mutations in CsaMLO8 confer resistance to the PM pathogen Podosphaera xanthii. Here, we used CRISPR/Cas9-mediated mutagenesis to generate PM resistance in the susceptible cucumber cv. 'Ilan'. Two transgene-free Csamlo8 CRISPR mutant lines (Csamlo-cr-1 and Csamlo-cr-2) were isolated, the first with a 5 bp deletion in exon 1, and the second harboring a 1280 bp deletion and 10 bp insertion between exons 1 and 5. Both lines showed high resistance to PM under semi-commercial growth conditions in the summer growing seasons of 2019 and 2021. These results provide the basis for generating transgene-free powdery mildew resistance in cucumber in any genetic background. This method can directly be employed on commercial cultivars and hybrid parental lines, and thereby substantially shorten and simplify the breeding process for PM resistance in cucumber.
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