D'Ann Rochon (1955-2022), a life of passion for plant virology.

D'Ann Rochon passed away on November 29th 2022. She is remembered for her outstanding contributions to the field of plant virology, her strong commitment to high quality science and her dedication to the training and mentorship of the next generation of scientists. She was a research scientist for Agriculture and Agri-Food Canada and an Adjunct Professor for the University of British Columbia. Her research program provided new insights on the infection cycle of tombusviruses and related viruses, including ground-breaking research on the structure of virus particles, the mechanisms of virus transmission by fungal zoospores, and the complexity of plant-virus interactions. She also developed diagnostic antibodies for plum pox virus and little cherry virus 2 that have had a significant impact on the management of these viruses.

A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV.

A spectrum of HRT-dependent hypersensitive responses elicited by the 52 amino acid N-terminus of turnip crinkle virus capsid protein and its mutants.

The capsid protein (CP) of turnip crinkle virus (TCV) is the elicitor of hypersensitive response (HR) and resistance mediated by the resistance protein HRT in the Di-17 ecotype of Arabidopsis. Here we identified the N-terminal 52-amino-acid R domain of TCV CP as the elicitor of HRT-dependent HR in Nicotiana benthamiana. Mutating this domain at position 6 (R6A), but not at positions 8 (R8A) or 14 (G14A), abolished HR in N. benthamiana. However, on Di-17 Arabidopsis leaves only R8A R domain elicited visible epidermal HR. When incorporated in infectious TCV RNAs, R8A and G14A mutations exerted dramatically different effects in Di-17 plants, as R8A caused systemic cell death whereas G14A led to complete restriction of the mutant virus. This continual spectrum of HR and resistance responses elicited by various R domain mutants suggests that the CP-HRT interaction could be perturbed by conformational changes in the R domain of TCV CP.

Generation of an Attenuated, Cross-Protective Pepino mosaic virus Variant Through Alignment-Guided Mutagenesis of the Viral Capsid Protein.

Mild variants of many viruses are able to protect infected plants from subsequent invasion by more severe variants of the same viruses through a process known as cross-protection. In the past, the cross-protective viral variants were commonly derived from mild field isolates that were sometimes genetically heterogeneous, providing variable levels of cross-protection. Here, we report a novel approach to rapidly generate cross-protective variants of the tomato-infecting Pepino mosaic virus (PepMV) independently of the availability of mild field isolates. Our approach sought to attenuate PepMV by mutating less conserved amino acid residues of the abundantly produced capsid protein (CP). These less-conserved amino acid residues were identified through multiple alignments of CPs of six potexviruses including PepMV, and were altered systematically to yield six PepMV mutants. These mutants were subsequently inoculated onto the model plant Nicotiana benthamiana, as well as tomato, to evaluate their accumulation levels, symptom severities, and cross-protection potentials. The mutant KD, in which the threonine (T) and alanine (A) residues at CP positions 66 and 67 were replaced with lysine (K) and aspartic acid (D), respectively, were found to accumulate to low levels in infected plants, cause very mild symptoms, and effectively protect both N. benthamiana and tomato against secondary infections by wild-type PepMV. These data suggest that our approach represents a simple, fast, and reliable way of generating attenuated viral variants capable of cross-protection.

Turnip crinkle virus coat protein inhibits the basal immune response to virus invasion in Arabidopsis by binding to the NAC transcription factor TIP.

Turnip crinkle virus (TCV) has been shown to interact with a NAC transcription factor, TIP, of Arabidopsis thaliana, via its coat protein (CP). This interaction correlates with the resistance response manifested in TCV-resistant Arabidopsis ecotype Di-17. We report that failure of a mutated CP to interact with TIP triggered the corresponding TCV mutant (R6A) to cause more severe symptoms in the TCV-susceptible ecotype Col-0. We hypothesized that TCV regulates antiviral basal immunity through TIP-CP interaction. Consistent with this hypothesis, we found that the rate of accumulation of R6A was measurably slower than wild-type TCV over the course of an infection. Notably, R6A was able to accumulate at similar rates as wild-type TCV in mutant plants with defects in salicylic acid (SA) signaling. Finally, plants with altered TIP expression provided evidence R6A's inability to evade the basal resistance response was likely associated with loss of ability for CP to bind TIP.

Triticum mosaic poacevirus enlists P1 rather than HC-Pro to suppress RNA silencing-mediated host defense.

Triticum mosaic virus (TriMV) is the type species of the newly established Poacevirus genus in the family Potyviridae. In this study, we demonstrate that in contrast to the helper component-proteinase (HC-Pro) of Potyvirus species, the P1 proteins of TriMV and Sugarcane streak mosaic poacevirus function in suppression of RNA silencing (SRS). TriMV P1 effectively suppressed silencing induced by single- or double-stranded RNAs (ss/ds RNAs), and disrupted the systemic spread of silencing signals at a step after silencing signal production. Interestingly, contrary to enhanced SRS activity of potyviral HC-Pro by co-expression with P1, the presence of TriMV HC-Pro reduced SRS activity of TriMV P1. Furthermore, TriMV P1 suppressed systemic silencing triggered by dsRNA more efficiently than the HC-Pro of Turnip mosaic potyvirus. Furthermore, TriMV P1 enhanced the pathogenicity of a heterologous virus. Our results established poaceviral P1 as a potent RNA silencing suppressor that probably employs a novel mechanism to suppress RNA silencing-based antiviral defense.

Virus infections in wild plant populations are both frequent and often unapparent.

PREMISE OF THE STUDY: Pathogens are thought to regulate host populations. In agricultural crops, virus infection reduces yield. However, in wild plants little is known about the spatial and temporal patterns of virus prevalence. Thus, pathogen effects on plant population dynamics are unclear. Prevalence data provide necessary background for (1) evaluating the effects of virus infection on plant population size and dynamics and (2) improving risk assessment of virus-resistant transgenic crops. METHODS: We used ELISA and RT-PCR to survey wild Cucurbita pepo populations over 4 years for five viruses, aphid-transmitted viruses of the genus Potyvirus as a group and PCR to survey for virus-resistance transgenes. In addition, we surveyed the literature for reports of virus prevalence in wild populations. KEY RESULTS: In 21 C. pepo populations, virus prevalence (0-74%) varied greatly among populations, years, and virus species. In samples analyzed by both ELISA and RT-PCR, RT-PCR detected 6-44% more viruses than did ELISA. Eighty percent of these infections did not cause any visually apparent symptoms. In our samples, the virus-resistance transgene was not present. In 30 published studies, 92 of 146 tested species were infected with virus, and infection rates ranged from 0.01-100%. Most published studies used ELISA, suggesting virus prevalence is higher than reported. CONCLUSIONS: In wild C. pepo, the demographic effects of virus are likely highly variable in space and time. Further, our literature survey suggests that such variation is probably common across plant species. Our results indicate that risk assessments for virus-resistant transgenic crops should not rely on visual symptoms or ELISA and should include data from multiple populations over multiple years.

Tritimovirus P1 functions as a suppressor of RNA silencing and an enhancer of disease symptoms.

Wheat streak mosaic virus (WSMV) is an eriophyid mite-transmitted virus of the genus Tritimovirus, family Potyviridae. Complete deletion of helper component-proteinase (HC-Pro) has no effect on WSMV virulence or disease synergism, suggesting that a different viral protein suppresses RNA silencing. RNA silencing suppression assays using Nicotiana benthamiana 16C plants expressing GFP were conducted with each WSMV protein; only P1 suppressed RNA silencing. Accumulation of GFP siRNAs was markedly reduced in leaves infiltrated with WSMV P1 at both 3 and 6 days post infiltration relative to WSMV HC-Pro and the empty vector control. On the other hand, helper component-proteinase (HC-Pro) of two species in the mite-transmitted genus Rymovirus, family Potyviridae was demonstrated to be a suppressor of RNA silencing. Symptom enhancement assays were conducted by inoculating Potato virus X (PVX) onto transgenic N. benthamiana. Symptoms produced by PVX were more severe on transgenic plants expressing WSMV P1 or potyvirus HC-Pro compared to transgenic plants expressing GFP or WSMV HC-Pro.

Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene.

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

The capsid protein of Turnip crinkle virus overcomes two separate defense barriers to facilitate systemic movement of the virus in Arabidopsis.

The capsid protein (CP) of Turnip crinkle virus (TCV) is a multifunctional protein needed for virus assembly, suppression of RNA silencing-based antiviral defense, and long-distance movement in infected plants. In this report, we have examined genetic requirements for the different functions of TCV CP and evaluated the interdependence of these functions. A series of TCV mutants containing alterations in the CP coding region were generated. These alterations range from single-amino-acid substitutions and domain truncations to knockouts of CP translation. The latter category also contained two constructs in which the CP coding region was replaced by either the cDNA of a silencing suppressor of a different virus or that of green fluorescent protein. These mutants were used to infect Arabidopsis plants with diminished antiviral silencing capability (dcl2 dcl3 dcl4 plants). There was a strong correlation between the ability of mutants to reach systemic leaves and the silencing suppressor activity of mutant CP. Virus particles were not essential for entry of the viral genome into vascular bundles in the inoculated leaves in the absence of antiviral silencing. However, virus particles were necessary for egress of the viral genome from the vasculature of systemic leaves. Our experiments demonstrate that TCV CP not only allows the viral genome to access the systemic movement channel through silencing suppression but also ensures its smooth egress by way of assembled virus particles. These results illustrate that efficient long-distance movement of TCV requires both functions afforded by the CP.

Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1.

Plant RNA silencing machinery enlists four primary classes of proteins to achieve sequence-specific regulation of gene expression and mount an antiviral defense. These include Dicer-like ribonucleases (DCLs), Argonaute proteins (AGOs), dsRNA-binding proteins (DRBs), and RNA-dependent RNA polymerases (RDRs). Although at least four distinct endogenous RNA silencing pathways have been thoroughly characterized, a detailed understanding of the antiviral RNA silencing pathway is just emerging. In this report, we have examined the role of four DCLs, two AGOs, one DRB, and one RDR in controlling viral RNA accumulation in infected Arabidopsis plants by using a mutant virus lacking its silencing suppressor. Our results show that all four DCLs contribute to antiviral RNA silencing. We confirm previous reports implicating both DCL4 and DCL2 in this process and establish a minor role for DCL3. Surprisingly, we found that DCL1 represses antiviral RNA silencing through negatively regulating the expression of DCL4 and DCL3. We also implicate DRB4 in antiviral RNA silencing. Finally, we show that both AGO1 and AGO7 function to ensure efficient clearance of viral RNAs and establish that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 and RDR6 favor less structured RNA targets. Our results resolve several key steps in the antiviral RNA silencing pathway and provide a basis for further in-depth analysis.

A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.

The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed.

Wheat streak mosaic virus Lacking Helper Component-Proteinase Is Competent to Produce Disease Synergism in Double Infections with Maize chlorotic mottle virus.

ABSTRACT The tritimovirus Wheat streak mosaic virus (WSMV) and the machlomovirus Maize chlorotic mottle virus (MCMV) each cause systemic chlorosis in infected maize plants. Infection of maize with both viruses produces corn lethal necrosis disease (CLND). Here, we report that complete deletion of the WSMV helper component-proteinase (HC-Pro) coding region had no effect on induction of CLND symptoms following coinoculation of maize with WSMV and MCMV. We further demonstrated that elevation of virus titers in double infections, relative to single infections, also was independent of WSMV HC-Pro. Thus, unlike potyvirus HC-Pro, WSMV HC-Pro was dispensable for disease synergism. Because disease synergism involving potyviruses requires HC-Pro-mediated suppression of posttranscriptional gene silencing (PTGS), we hypothesized that WSMV HC-Pro may not be a suppressor of PTGS. Indeed, WSMV HC-Pro did not suppress PTGS of a green fluorescent protein (GFP) transgene in an Agrobacterium-mediated coinfiltration assay in which potyvirus HC-Pro acted as a strong suppressor. Furthermore, coinfiltration with potyvirus HC-Pro, but not WSMV HC-Pro, resulted in elevated levels of the GFP target mRNA under conditions which trigger PTGS. Collectively, these results revealed significant differences in HC-Pro function among divergent genera of the family Potyviridae and suggest that the tritimovirus WSMV utilizes a gene other than HC-Pro to suppress PTGS and mediate synergistic interactions with unrelated viruses.

RDR6 has a broad-spectrum but temperature-dependent antiviral defense role in Nicotiana benthamiana.

SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from Arabidopsis thaliana, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the Nicotiana benthamiana homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested and that this effect was more pronounced at higher growth temperatures. Diminished expression of NbRDR6 also permitted efficient multiplication of tobacco mosaic virus in the shoot apices, leading to serious disruption with microRNA-mediated developmental regulation. Based on these results, we propose that NbRDR6 participates in the antiviral RNA silencing pathway that is stimulated by rising temperatures but suppressed by virus-encoded silencing suppressors. The relative strengths of these two factors, along with other plant defense components, critically influence the outcome of virus infections.

Suppressors of RNA silencing encoded by plant viruses and their role in viral infections.

RNA silencing as a robust host defense mechanism against plant viruses is generally countered by virus-encoded silencing suppressors. This strategy is now increasingly recognized to be used by animal viruses as well. We present here an overview of the common features shared by some of the better studied plant viral silencing suppressors. We then briefly describe the characteristics of the few reported animal viral suppressors, notably their extraordinary ability of cross-kingdom suppression. We next discuss the basis for biased protection of viral RNA and subviral parasites by silencing suppressors, the link between movement and silencing suppression, the influence of temperature on the outcome of viral infection and the effect of viral silencing suppressors on the microRNA pathway.

The nuclear localization of the Arabidopsis transcription factor TIP is blocked by its interaction with the coat protein of Turnip crinkle virus.

We have previously reported that TIP, an Arabidopsis protein, interacts with the coat protein (CP) of Turnip crinkle virus (TCV) in yeast cells and that this interaction correlated with the resistance response in the TCV-resistant Arabidopsis ecotype Dijon-17. TIP was also able to activate transcription of reporter genes in yeast cells, suggesting that it is likely a transcription factor. We have now verified the physical interaction between TIP and TCV CP in vitro and showed that CP mutants unable to interact with TIP in yeast cells bind TIP with much lower affinity in vitro. Secondly, we have performed gel shift experiments demonstrating that TIP does not bind to DNA in a sequence-specific manner. The subcellular localization of TIP was also investigated by transiently expressing green fluorescence protein (GFP)-tagged TIP in Nicotiana benthamiana plant cells, which showed that GFP-tagged TIP localizes primarily to nuclei. Significantly, co-expression of TCVCP and GFP-TIP prevented the nuclear localization of TIP. Together, these results suggest that TIP might be a transcription factor involved in regulating the defense response of Arabidopsis to TCV and that its normal role is compromised by interaction with the invading viral CP.

RNA silencing-suppressor function of Turnip crinkle virus coat protein cannot be attributed to its interaction with the Arabidopsis protein TIP.

The interaction of the coat protein (CP) of Turnip crinkle virus (TCV) with a host protein, TCV-interacting protein (TIP), from Arabidopsis thaliana has been reported previously. This interaction correlates with the ability of TCV CP to elicit the resistance response that is mediated by the resistance gene HRT in Arabidopsis ecotype Di-17. It has also been established that TCV CP is a suppressor of RNA silencing, a process by which the host plant targets viral RNA for degradation. These results have led to the speculation that TIP might be a component of the RNA-silencing pathway and that TCV CP suppresses RNA silencing through its interaction with TIP. In the current report, a number of TCV CP mutants have been investigated for their ability to suppress RNA silencing. These mutants include single amino acid substitution mutants that are known to have lost their ability to interact with TIP, as well as deletion mutants of TCV CP that are of different sizes and from different regions of the protein. Results showed that each of the single amino acid substitution mutants tested retained high levels of RNA silencing-suppressor activity. In addition, a mutant containing a 5 aa deletion in the region that is known to be critical for TIP interaction retained the ability to suppress RNA silencing significantly. Larger deletions in all regions of TCV CP abolished silencing-suppressor activity. It can be concluded from these results that the RNA silencing-suppressor activity of TCV CP cannot be attributed to its ability to interact directly with TIP.

The coat protein of turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step.

Posttranscriptional gene silencing (PTGS), or RNA silencing, is a sequence-specific RNA degradation process that targets foreign RNA, including viral and transposon RNA for destruction. Several RNA plant viruses have been shown to encode suppressors of PTGS in order to survive this host defense. We report here that the coat protein (CP) of Turnip crinkle virus (TCV) strongly suppresses PTGS. The Agrobacterium infiltration system was used to demonstrate that TCV CP suppressed the local PTGS as strongly as several previously reported virus-coded suppressors and that the action of TCV CP eliminated the small interfering RNAs associated with PTGS. We have also shown that the TCV CP must be present at the time of silencing initiation to be an effective suppressor. TCV CP was able to suppress PTGS induced by sense, antisense, and double-stranded RNAs, and it prevented systemic silencing. These data suggest that TCV CP functions to suppress RNA silencing at an early initiation step, likely by interfering the function of the Dicer-like RNase in plants.

Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing.

Tomato bushy stunt virus (TBSV) is one of few RNA plant viruses capable of moving systemically in some hosts in the absence of coat protein (CP). TBSV also encodes another protein (p19) that is not required for systemic movement but functions as a symptom determinant in Nicotiana benthamiana. Here, the role of both CP and p19 in the systemic spread has been reevaluated by utilizing transgenic N. benthamiana plants expressing the movement protein (MP) of Red clover necrotic mosaic virus and chimeric TBSV mutants that express CP of Turnip crinkle virus. Through careful examination of the infection phenotype of a series of mutants with changes in the CP and p19 genes, we demonstrate that both of these genes are required for efficient systemic invasion of TBSV in N. benthamiana. The CP likely enables efficient viral unloading from the vascular system in the form of assembled virions, whereas p19 enhances systemic infection by suppressing the virus-induced gene silencing.