Synergistic interactions of begomoviruses with Sweet potato chlorotic stunt virus (genus Crinivirus) in sweet potato (Ipomoea batatas L.)

Three hundred and ninety‐four sweet potato accessions from Latin America and East Africa were screened by polymerase chain reaction (PCR) for the presence of begomoviruses, and 46 were found to be positive. All were symptomless in sweet potato and generated leaf curling and/or chlorosis in Ipomoea setosa. The five most divergent isolates, based on complete genome sequences, were used to study interactions with Sweet potato chlorotic stunt virus (SPCSV), known to cause synergistic diseases with other viruses. Co‐infections led to increased titres of begomoviruses and decreased titres of SPCSV in all cases, although the extent of the changes varied notably between begomovirus isolates. Symptoms of leaf curling only developed temporarily in combination with isolate StV1 and coincided with the presence of the highest begomovirus concentrations in the plant. Small interfering RNA (siRNA) sequence analysis revealed that co‐infection of SPCSV with isolate StV1 led to relatively increased siRNA targeting of the central part of the SPCSV genome and a reduction in targeting of the genomic ends, but no changes to the targeting of StV1 relative to single infection of either virus. These changes were not observed in the interaction between SPCSV and the RNA virus Sweet potato feathery mottle virus (genus Potyvirus), implying specific effects of begomoviruses on RNA silencing of SPCSV in dually infected plants. Infection in RNase3‐expressing transgenic plants showed that this protein was sufficient to mediate this synergistic interaction with DNA viruses, similar to RNA viruses, but exposed distinct effects on RNA silencing when RNase3 was expressed from its native virus, or constitutively from a transgene, despite a similar pathogenic outcome.     

Transgenic <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> plants resistant to cucumber green mottle mosaic virus based on RNA silencing

RNA silencing technology was used to confer resistance to cucumber green mottle mosaic virus (CGMMV). Nicotiana benthamiana was transformed with a transgene designed to produce an inverted repeat RNA containing CGMMV-coat protein gene (CP) sequences, which were separated by an intron sequence, under the control of the cauliflower mosaic virus 35S promoter. We attempted to confirm the resistance of seven independent transgenic lines; five lines showed resistance to CGMMV infection. The systemic spread of virus was prevented after the inoculation of CGMMV, and the CP-specific short interfering RNA (siRNA) was detected in resistant lines. Thus, the resistance against CGMMV through RNA silencing is strong and efficient.     

Viral class 1 RNase III involved in suppression of RNA silencing

Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.     

Development of FRET-based high-throughput screening for viral RNase III inhibitors

The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose–response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.     

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.     

Specificity of the effect of sap-transmissible viruses in increasing the accumulation of luteoviruses in co-infected plants

The concentration of potato leafroll luteovirus (PLRV) (c. 1300 ng/g leaf) in singly infected Nicotiana clevelandii plants was increased up to 10-fold in plants co-infected with each of several potyviruses, or with narcissus mosaic potexvirus, carrot mottle virus or each of three tobravirus isolates. With the tobraviruses, PLRV concentration was increased equally by co-infection with either NM-type isolates (coat protein-free cultures containing RNA-1) or M-type isolates (particle-producing cultures containing RNA-1 and RNA-2). In contrast, the accumulation of PLRV was not substantially affected by co-infection with either of two nepoviruses, cucumber mosaic cucumovirus, broad bean mottle bromovirus, alfalfa mosaic virus, pea enation mosaic virus or parsnip yellow fleck virus. The specificity of these interactions between PLRV and sap-transmissible viruses was retained in tests made in Nicotiana benthamiana and when beet western yellows luteovirus was used instead of PLRV.     

A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13

For some Cas nucleases, trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection. Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture. Here, we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool. We systematically characterized AsCas12a, LbCas12a, LwaCas13a, and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection. Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input. Using this tool, we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field. Our method, from sample preparation to result readout, could be rapidly and easily deployed in the field. This system could be extended to other crop pathogens, including those that currently lack a detection method and have metabolite profiles that make detection challenging. This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping, transgene detection, and qualitative detection of gene expression in the field.

     

Resistance to sweet potato virus disease (SPVD) in wild East African Ipomoea

Sweet potato virus disease (SPVD), the most harmful disease of sweet potatoes in East Africa, is caused by mixed infection with sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Wild Ipomoea spp. native to East Africa (J cairica, I. hildebrandtii, I. involucra and J wightii) were graft-inoculated with SPVD-affected sweet potato scions. Inoculated plants were monitored for symptom development and tested for SPFMV and SPCSV by grafting to the indicator plant J setosa, and by enzyme-linked immunosorbent assay (ELISA). Virus-free scions of sweet potato cv. Jersey were grafted onto these wild Ipomoea spp. in the field, and scions collected 3 wk later were rooted in the greenhouse and tested for viruses using serological tests and bioassays. In all virus tests, J cairica and J involucra were not infected with either SPFMV or SPCSV. J wightii was infected with SPFMV, but not SPCSV, in the field and following experimental inoculation; J hildebrandtii was infected with SPCSV, but not SPFMV, following experimental inoculation. These data provide the first evidence of East African wild Ipomoea germplasm resistant to the viruses causing SPVD.     

Aspects of resistance to sweet potato virus disease in sweet potato

In field trials during the first and the second rainy season of 1996 in Uganda, whiteflies were similarly abundant and aphids were absent on three clones of sweet potato (NIS-93–63, cv. Tanzania and cv. New Kawogo) although the three clones differed considerably in their resistance to sweet potato virus disease (SPVD), a complex disease resulting from infection by both the aphid-borne sweet potato feathery mottle virus (SPFMV) and the whitefly-borne sweet potato chlorotic stunt virus (SPCSV). This suggests that vector resistance does not determine the relative SPVD resistance of these genotypes. SPFMV alone had only a low virus titre in sweet potato cvs Tanzania and New Kawogo, became increasingly difficult to detect in plants of these cultivars and was seldom acquired by aphids. However, this resistance to SPFMV was not apparent in plants which were also infected with SPCSV. Plants then had a high SPFMV titre, appeared unable to eliminate SPFMV and provided good sources for aphids to acquire it.     

A critical domain of Sweet potato chlorotic fleck virus nucleotide‐binding protein (NaBp) for RNA silencing suppression,nuclear localization and viral pathogenesis

RNA silencing is an important mechanism of antiviral defence in plants. To counteract this resistance mechanism, many viruses have evolved RNA silencing suppressors. In this study, we analysed five proteins encoded by Sweet potato chlorotic fleck virus (SPCFV) for their abilities to suppress RNA silencing using a green fluorescent protein (GFP)‐based transient expression assay in Nicotiana benthamiana line 16c plants. Our results showed that a putative nucleotide‐binding protein (NaBp), but not other proteins encoded by the virus, could efficiently suppress local and systemic RNA silencing induced by either sense or double‐stranded RNA (dsRNA) molecules. Deletion mutation analysis of NaBp demonstrated that the basic motif (an arginine‐rich region) was critical for its RNA silencing suppression activity. Using confocal laser scanning microscopy imaging of transfected protoplasts expressing NaBp fused to GFP, we showed that NaBp accumulated predominantly in the nucleus. Mutational analysis of NaBp demonstrated that the basic motif represented part of the nuclear localization signal. In addition, we demonstrated that the basic motif in NaBp was a pathogenicity determinant in the Potato virus X (PVX) heterogeneous system. Overall, our results demonstrate that the basic motif of SPCFV NaBp plays a critical role in RNA silencing suppression, nuclear localization and viral pathogenesis.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

Identification of Pns12 as the second silencing suppressor of <Emphasis Type="Italic">Rice gall dwarf virus</Emphasis>

RNA silencing is a conserved mechanism found ubiquitously in eukaryotic organisms. It has been used to regulate gene expression and development. In addition, RNA silencing serves as an important mechanism in plants’ defense against invasive nucleic acids, such as viruses, transposons, and transgenes. As a counter-defense, most plants, and some animal viruses, encode RNA silencing suppressors to interfere at one or several points of the silencing pathway. In this study, we showed that Pns12 of RGDV (Rice gall dwarf virus) exhibits silencing suppressor activity on the reporter green fluorescent protein in transgenic Nicotiana benthamiana line 16c. Pns12 of RGDV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Expression of Pns12 also enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, these results suggested that RGDV Pns12 functions as a virus suppressor of RNA silencing, which might target an upstream step of dsRNA formation in the RNA silencing pathway. Furthermore, we showed that Pns12 is localized mainly in the nucleus of N. benthamiana leaf cells.     

Mitogen‐activated protein kinase phosphatase 1 reduces the replication efficiency of Bamboo mosaic virus in Nicotiana benthamiana

In plants, the mitogen‐activated protein kinase (MAPK) cascades are the central signaling pathways of the complicated defense network triggered by the perception of pathogen‐associated molecular patterns to repel pathogens. The Arabidopsis thaliana MAPK phosphatase 1 (AtMKP1) negatively regulates the activation of MAPKs. Recently, the AtMKP1 homolog of Nicotiana benthamiana (NbMKP1) was found in association with the Bamboo mosaic virus (BaMV) replication complex. This study aimed to investigate the role of NbMKP1 in BaMV multiplication in N. benthamiana. Silencing of NbMKP1 increased accumulations of the BaMV‐encoded proteins and the viral genomic RNA, although the same condition reduced the infectivity of Pseudomonas syringae pv. tomato DC3000 in N. benthamiana. On the other hand, overexpression of NbMKP1 decreased the BaMV coat protein accumulation in a phosphatase activity‐dependent manner in protoplasts. NbMKP1 also negatively affected the in vitro RNA polymerase activity of the BaMV replication complex. Collectively, the activity of NbMKP1 seems to reduce BaMV multiplication, inconsistent with the negatively regulatory role of MKP1 in MAPK cascades in terms of warding off fungal and bacterial invasion. In addition, silencing of NbMKP1 increased the accumulation of Foxtail mosaic virus but decreased Potato virus X. The discrepant effects exerted by NbMKP1 on different pathogens foresee the difficulty to develop plants with broad‐spectrum resistance through genetically manipulating a single player in MAPK cascades.     

RNA silencing suppressor activity of Velvet bean severe mosaic begomovirus DNA-A encoded proteins

Velvet bean severe mosaic virus (VbSMV) is a bipartite DNA virus infecting Mucuna pruriens (Velvet bean), belongs to the genus Begomovirus in the family Geminiviridae. Velvet bean is a medicinal plant of enormous medicinal value. In the present study, it was delineated that proteins encoded by VbSMV viz. AV2 (pre-coat protein), AC2 (TrAP), AV1 (coat protein) are suppressors of RNA silencing as identified through Agrobacterium co-infiltration assays using Nicotiana benthamiana as a host plant. AV2 showed strong suppressor activity whereas AC1 and AV1 were found to be weak suppressors. To the best of our knowledge, this is the first report on identification of suppressor of RNA silencing encoded by VbSMV infecting a medicinal plant.     

Expression of an extracellular ribonuclease gene increases resistance to Cucumber mosaic virus in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

     

Generation of virus‐resistant potato plants by RNA genome targeting

CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA‐targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sgRNA construct in the plants. Our data further demonstrate that appropriately designed sgRNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.