Rapid selection of potyviral cross-protection effective mutants from the local lesion host after nitrous acid mutagenesis.

Zucchini yellow mosaic virus (ZYMV) seriously damages cucurbits worldwide. Control of ZYMV by cross-protection has been practised for decades, but selecting useful mild viruses is time-consuming and laborious. Most attenuated potyviruses used for cross-protection do not induce hypersensitive reaction (HR) in Chenopodium quinoa, a local lesion host Chenopodium quinoa. Here, severe ZYMV TW-TN3 tagged with green fluorescent protein (GFP), designated ZG, was used for nitrous acid mutagenesis. From three trials, 11 mutants were identified from fluorescent spots without HR in inoculated C. quinoa leaves. Five mutants caused attenuated symptoms in squash plants. The genomic sequences of these five mutants revealed that most of the nonsynonymous changes were located in the HC-Pro gene. The replacement of individual mutated HC-Pros in the ZG backbone and an RNA silencing suppression (RSS) assay indicated that each mutated HC-Pro is defective in RSS function and responsible for reduced virulence. Four mutants provided high degrees of protection (84%-100%) against severe virus TW-TN3 in zucchini squash plants, with ZG 4-10 being selected for removal of the GFP tag. After removal of the GFP gene, Z 4-10 induced symptoms similar to ZG 4-10 and still provided 100% protection against TW-TN3 in squash, thus is considered not a genetically engineered mutant. Therefore, using a GFP reporter to select non-HR mutants of ZYMV from C. quinoa leaves is an efficient way to obtain beneficial mild viruses for cross-protection. This novel approach is being applied to other potyviruses.

Modification of the N-terminal FWKG-αH1 element of potyviral HC-Pro affects its multiple functions and generates effective attenuated mutants for cross-protection.

Control of plant viruses by cross-protection is limited by the availability of effective protective strains. Incorporation of an NIa-protease processing site in the extreme N-terminal region of the helper component protease (HC-Pro) of turnip mosaic virus (TuMV) resulted in a mutant virus TuHND I that induced highly attenuated symptoms. Recombination analysis verified that two variations, F7I mutation and amino acid 7-upstream-deletion, in HC-Pro co-determined TuHND I attenuation. TuHND I provided complete protection to Nicotiana benthamiana and Brassica campestris subsp. chinensis plants against infection by the severe parental strain. Aphid transmission tests revealed that TuHND I was not aphid-transmissible. An RNA silencing suppression (RSS) assay by agroinfiltration suggested the RSS-defective nature of the mutant HC-Pro. In the context (amino acids 3-17) encompassing the two variations of HC-Pro, we uncovered an FWKG-α-helix 1 (αH1) element that influenced the functions of aphid transmission and RSS, whose motifs were located far downstream. We further demonstrated that HC-Pro F7 was a critical residue on αH1 for HC-Pro functions and that reinstating αH1 in the RSS-defective HC-Pro of TuHND I restored the protein's RSS function. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated the FWKG-αH1 element as an integral part of the HC-Pro self-interaction domain. The possibility of regulation of the mechanistically independent functions of RSS and aphid transmission by the FWKG-αH1 element is discussed. Extension of TuMV HC-Pro FWKG-αH1 variations to another potyvirus, zucchini yellow mosaic virus, also generated nonaphid-transmissible cross-protective mutant viruses. Hence, the modification of the FWKG-αH1 element can generate effective attenuated viruses for the control of potyviruses by cross-protection.

Targeting of cucumber necrosis virus coat protein to the chloroplast stroma attenuates host defense response.

Cucumber necrosis virus (CNV) is a (+)ssRNA virus that elicits spreading local and systemic necrosis in Nicotiana benthamiana. We previously showed that the CNV coat protein (CP) arm functions as a chloroplast transit peptide that targets a CP fragment containing the S and P domains to chloroplasts during infection. Here we show that several CP arm mutants that inefficiently target chloroplasts, along with a mutant that lacks the S and P domains, show an early onset of more localized necrosis along with protracted induction of pathogenesis related protein (PR1a). Agroinfiltrated CNV CP is shown to interfere with CNV p33 and Tomato bushy stunt virus p19 induced necrosis. Additionally, we provide evidence that a CP mutant that does not detectably enter the chloroplast stroma induces relatively higher levels of several plant defense-related genes compared to WT CNV. Together, our data suggest that targeting of CNV CP to the chloroplast stroma interferes with chloroplast-mediated plant defense.

Modulation of disease severity by plant positive-strand RNA viruses: The complex interplay of multifunctional viral proteins, subviral RNAs and virus-associated RNAs with plant signaling pathways and defense responses.

Plant viruses induce a range of symptoms of varying intensity, ranging from severe systemic necrosis to mild or asymptomatic infection. Several evolutionary constraints drive virus virulence, including the dependence of viruses on host factors to complete their infection cycle, the requirement to counteract or evade plant antiviral defense responses and the mode of virus transmission. Viruses have developed an array of strategies to modulate disease severity. Accumulating evidence has highlighted not only the multifunctional role that viral proteins play in disrupting or highjacking plant factors, hormone signaling pathways and intracellular organelles, but also the interaction networks between viral proteins, subviral RNAs and/or other viral-associated RNAs that regulate disease severity. This review focusses on positive-strand RNA viruses, which constitute the majority of characterized plant viruses. Using well-characterized viruses with different genome types as examples, recent advances are discussed as well as knowledge gaps and opportunities for further research.

Satellite RNAs interfere with the function of viral RNA silencing suppressors.

Viral satellite RNAs (satRNAs) are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs), Cucumber mosaic virus (CMV) 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA)-induced silencing of a ß-glucuronidase (GUS) gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat) via the Y-Sat-derived small interfering RNAs (siRNAs), which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function.