The polypeptide extract of the dry mycelium of Penicillium chrysogenum (PDMP) can protect tobacco plants from tobacco mosaic virus (TMV), although the potential mechanism underlying PDMP-mediated TMV resistance remains unknown. In our study, we analyzed this potential mechanism via RNA sequencing (RNA-seq) and found that the ABA biosynthetic pathway and β-1,3-glucanase, a callose-degrading enzyme, might play an important role in the PDMP-induced priming of resistance to TMV. To test our hypothesis, we successfully generated a Nicotiana benthamiana ABA biosynthesis mutant and evaluated the role of the ABA pathway in PDMP-induced callose deposition during plasmodesmata resistance to TMV infection via aniline blue staining, quantitative real-time polymerase chain reaction, western blotting, and immunoelectron microscopy. Our results suggested that PDMP can induce callose priming to defend against TMV movement. PDMP inhibited TMV movement by increasing callose deposition around plasmodesmata, but this phenomenon did not occur in the ABA biosynthesis mutant; moreover, these effects of PDMP on callose deposition could be rescued by treatment with exogenous ABA. Our results suggested that callose deposition around plasmodesmata in wild-type plants is mainly responsible for the restriction of TMV movement during the PDMP-induced defensive response to TMV infection and that ABA biosynthesis apparently plays a crucial role in PDMP-induced callose priming for enhancing defense against TMV.

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Quantitative real-time PCR (RT-qPCR) is a widely used method for studying alterations in gene expression upon infections caused by diverse pathogens such as viruses. Positive-sense single-stranded (ss(+)) RNA viruses form a major part of all known plant viruses, and some of them are damaging pathogens of agriculturally important crops. Analysis of gene expression following infection by ss(+) RNA viruses is crucial for the identification of potential anti-viral factors. However, viral infections are known to globally affect gene expression and therefore selection and validation of reference genes for RT-qPCR is particularly important. In this study, the expression of commonly used reference genes for RT-qPCR was studied in Nicotiana benthamiana following single infection by 11 ss(+) RNA viruses, including five tobamoviruses, four potyviruses, one potexvirus and one polerovirus. Stability of gene expression was analyzed in parallel by four commonly used algorithms: geNorm, NormFinder, BestKeeper, and Delta CT, and RefFinder was finally used to summarize all the data. The most stably expressed reference genes differed significantly among the viruses, even when those viruses were from the same genus. Our study highlights the importance of the selection and validation of reference genes upon different viral infections.

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An isolate of chilli veinal mottle virus (ChiVMV; genus Potyvirus) of Solanum nigrum L. from southwest China (ChiVMV-YunN/Yuxi) was identified and sequenced (GenBank: OP404087). Comparison with other ChiVMV isolates and recombination analyses suggested a recombinant origin. The most significant recombination event among all 21 complete ChiVMV isolates was an ending breakpoint at 1408–1488 for ChiVMV-YunN/Yuxi with ChiVMV-TaiW and ChiVMV-YunN/Ca operating as the respective major and minor parents. Interestingly, the 5′ UTR of ChiVMV-YunN/Yuxi is 15 nucleotides (‘AAAAATAAAACAACC’) longer than other reported isolates. A full-length clone of ChiVMV-YunN/Yuxi was constructed and was shown to be infectious in Nicotiana benthamiana. The additional 15 nt of 5′ UTR in ChiVMV-YunN/Yuxi was stable when transmitted through three generations. Experiments with modified clones showed that the additional 15 nt are essential for infection by this isolate.