Efficient artificial microRNA-mediated resistance against zucchini yellow mosaic virus in zucchini via agroinfiltration.

Zucchini yellow mosaic virus (ZYMV) infects cucurbits causing yellow mosaic in leaves, malformations in fruits, and degradation of the product quality. RNA interference (RNAi) is a cellular mechanism in eukaryotes and it is exploited to protect them against viruses. The artificial micro RNA (amiRNA) mediated approach was employed to develop resistance against ZYMV. Four amiRNAs, amiZYMV_HC-115s and amiZYMV_HC-1162s (sense), amiZYMV_HC-182as and amiZYMV_HC-196as (antisense), were computationally designed and introduced into the AtMIR390a backbone. At four days post agroinfiltration (dpa) of zucchini cotyledons the corresponding pre- and the mature amiRNAs were identified in local tissue. Upon ZYMV inoculation of zucchini, ZYMV titer was significantly lower where amiZYMV_HCs were applied in relation to control starting at two days post inoculation (dpi). Control zucchini plants exhibited symptoms at 5-8 dpi, whereas the amiZYMV_HC-treated zucchini had symptoms at 14 dpi; at 21 dpi treated zucchini exhibited a 16 %, 19 %, 32 %, and 42.5 % protection, respectively. For luffa, we observed a lower protection (0 %, 17 %, 22.5 %, and 31 % at 21 dpi). Nicotiana benthamiana DCL4 knock-down mutants were infected by ZYMV, whereas when the amiZYMV_HC-196as was agroinfiltrated ZYMV was not detected by RT-PCR. These results indicate that amiRNA-mediated resistance could be applied against ZYMV in zucchini.

DsRNA-mediated protection against two isolates of Papaya ringspot virus through topical application of dsRNA in papaya.

Papaya ringspot virus (PRSV) infections in papaya result in heavy yield losses, severely affecting the papaya industry worldwide, and hence warranting for effective control measures. In the past, transgenic papaya cultivars were developed that overexpressed parts of the PRSV genome and exhibited high levels of virus resistance. In the present study, a non-transgenic approach was employed, in which in vitro produced dsRNA molecules derived from a PRSV isolate from South India (PRSV-Tirupati) was tested for dsRNA-mediated protection against two isolates of PRSV through topical application of the dsRNA on papaya. The results showed that the dsRNA molecules from both the coat protein (CP) and helper component-proteinase (HC-Pro) genes of the PRSV-Tirupati isolate conferred 100 % resistance against PRSV-Tirupati infection. Further, the same dsRNA molecules were highly effective against the PRSV-Delhi isolate on the papaya cv. Pusa Nanha, conferring a resistance of 94 % and 81 %, respectively. Systemic papaya leaves of the dsRNA-treated plants were virus-free at 14 days post-inoculation, confirming the robustness of this non-transgenic virus control strategy. In contrast, the control TMV dsRNA did not protect against the PRSV infection. This study on the topical application of dsRNA opened up a new avenue for the control of papaya ringspot disease worldwide.

RNA-based vaccination of Bhut Jolokia pepper (Capsicum chinense Jacq.) against cucumber mosaic virus.

Cucumber mosaic virus (CMV) causes great losses in Bhut Jolokia pepper (Capsicum chinense Jacq.) plantations in Assam, India. To investigate possible means to induce plant resistance against this virus, the crude extract of bacterially-expressed double-stranded (ds) RNA, derived from CMV-2b gene (dsRNA_CMV-2b), was exogenously applied along with CMV-G strain onto Bhut Jolokia plants. In this 'RNA-vaccination' bioassay, disease incidence, assessed by testing the plants at 21 days post inoculation by DAS-ELISA, ranged from 0 to 29% in case of dsRNA-treated plants, and from 55 to 92% when only CMV was applied. CMV-infected pepper plants became severely stunted, having dull light green foliage with leathery appearance, whereas plants receiving dsRNA_CMV-2b exhibited milder symptoms or remained healthy. The results obtained suggest that this non-transgenic approach has a considerable effect in protecting pepper against CMV.

Exogenously applied dsRNA molecules deriving from the Zucchini yellow mosaic virus (ZYMV) genome move systemically and protect cucurbits against ZYMV.

Zucchini yellow mosaic virus (ZYMV) causes serious damage in a large number of cucurbits, and control measures are necessary. Transgenic cucurbits expressing parts of the ZYMV genome have been shown to be resistant to the cognate virus. A non-transgenic approach involving the exogenous application of double-stranded RNA (dsRNA) has also been shown to induce resistance in tobacco against Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV). In the present study, dsRNA molecules derived from the helper component-proteinase (HC-Pro) and coat protein (CP) genes of the ZYMV_DE_2014 isolate were produced in vitro. On exogenous dsRNA application in cucumber, watermelon and squash plants, dsRNA HC-Pro conferred resistance of 82%, 50% and 18%, and dsRNA CP molecules of 70%, 43% and 16%, respectively. On deep sequencing analysis of ZYMV-infected watermelon, hot-spot regions for viral small interfering RNAs (vsiRNAs) in the genome of ZYMV were identified. Stem-loop reverse transcription-polymerase chain reaction (RT-PCR) detection of selected 21-nucleotide-long vsiRNAs in plants that received only dsRNA molecules suggested that the dsRNAs exogenously applied onto plants were successfully diced, thus initiating RNA silencing. dsRNA molecules were found to be progressively degraded in planta, and strongly detected by semi-quantitative RT-PCR for at least 9 days after exogenous application. Moreover, dsRNA molecules were detected in systemic tissue of watermelon and squash, showing that dsRNA is transported long distances in these plants.

Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco.

MAIN CONCLUSION: External application of dsRNA molecules from Tobacco mosaic virus (TMV) p126 and CP genes confers significant resistance against TMV infection. Exogenously applied dsRNA exhibits a rapid systemic trafficking in planta , and it is processed successfully by DICER-like proteins producing small interfering RNAs. RNA interference (RNAi) is a sequence-specific, post-transcriptional gene silencing mechanism, induced by double-stranded RNA (dsRNA), which protects eukaryotic cells against invasive nucleic acids like viruses and transposons. In the present study, we used a non-transgenic strategy to induce RNAi in Nicotiana tabacum cv. Xanthi plants against TMV. DsRNA molecules for the p126 (TMV silencing suppressor) and coat protein (CP) genes were produced by a two-step PCR approach followed by in vitro transcription. The application of TMV p126 dsRNA onto tobacco plants induced greater resistance against TMV infection as compared to CP dsRNA (65 vs. 50 %). This study also reported the fast systemic spread of TMV p126 dsRNA from the treated (local) to non-treated (systemic) leaves beginning from 1 h post-application, confirmed by both conventional and real-time RT-PCR. Furthermore, we employed a stem-loop RT-PCR and confirmed the presence of a putative viral siRNA for up to 9 days in local leaves and up to 6 days in systemic leaves post-application. The approach employed could represent a simple and environmentally safe way for the control of plant viruses in future agriculture.