Sumoylation of Turnip mosaic virus RNA Polymerase Promotes Viral Infection by Counteracting the Host NPR1-Mediated Immune Response.

Sumoylation is a transient, reversible dynamic posttranslational modification that regulates diverse cellular processes including plant-pathogen interactions. Sumoylation of NPR1, a master regulator of basal and systemic acquired resistance to a broad spectrum of plant pathogens, activates the defense response. Here, we report that NIb, the only RNA-dependent RNA polymerase of Turnip mosaic virus (TuMV) that targets the nucleus upon translation, interacts exclusively with and is sumoylated by SUMO3 (SMALL UBIQUITIN-LIKE MODIFIER3), but not the three other Arabidopsis thaliana SUMO paralogs. TuMV infection upregulates SUMO3 expression, and the sumoylation of NIb by SUMO3 regulates the nuclear-cytoplasmic partitioning of NIb. We identified the SUMO-interacting motif in NIb that is essential for its sumoylation and found that knockout or overexpression of SUMO3 suppresses TuMV replication and attenuates viral symptoms, suggesting that SUMO3 plays dual roles as a host factor of TuMV and as an antiviral defender. Sumoylation of NIb by SUMO3 is crucial for its role in suppressing the host immune response. Taken together, our findings reveal that sumoylation of NIb promotes TuMV infection by retargeting NIb from the nucleus to the cytoplasm where viral replication takes place and by suppressing host antiviral responses through counteracting the TuMV infection-induced, SUMO3-activated, NPR1-mediated resistance pathway.

Heterotrimeric G-proteins facilitate resistance to plant pathogenic viruses in Arabidopsis thaliana (L.) Heynh.

Heterotrimeric G-proteins, consisting of Gα, Gß and Gγ subunits, are important signal transducers in eukaryotes. In plants, G-protein-mediated signaling contributes to defense against a range of fungal and bacterial pathogens. Here we studied response of G-protein-deficient mutants to ssRNA viruses representing 2 different families: Cucumber mosaic virus (CMV) (Bromoviridae) and Turnip mosaic virus (TuMV) (Potyviridae). We found that development of spreading necrosis on infected plants was suppressed in the Gß-deficient mutant (agb1-2) compared to wild type and Gα-deficient mutant (gpa1-4). In accordance, ion leakage caused by viral infection was also significantly reduced in agb1-2 compared to wild type and gpa1-4. Nevertheless, both viruses replicated better in agb1-2 plants, while gpa1-4 was similar to wild type. Analysis of pathogenesis-related genes showed that Gß negatively regulated salicylic acid, jasmonic acid and abscisic acid marker genes during CMV and TuMV infections. Interestingly, analysis of salicylic acid deficient transgenic plants indicated that salicylic acid did not affect resistance against these viruses and did not influence the Gß-mediated defense response. We conclude that heterotrimeric G-proteins play a positive role in defense against viral pathogens probably by promoting cell death.

Development of a Mild Viral Expression System for Gain-Of-Function Study of Phytoplasma Effector In Planta.

PHYL1 and SAP54 are orthologs of pathogenic effectors of Aster yellow witches'-broom (AYWB) phytoplasma and Peanut witches'-broom (PnWB) phytoplasma, respectively. These effectors cause virescence and phyllody symptoms (hereafter leafy flower) in phytoplasma-infected plants. T0 lines of transgenic Arabidopsis expressing the PHYL1 or SAP54 genes (PHYL1 or SAP54 plants) show a leafy flower phenotype and result in seedless, suggesting that PHYL1 and SAP54 interfere with reproduction stage that restrict gain-of-function studies in the next generation of transgenic plants. Turnip mosaic virus (TuMV) mild strain (TuGK) has an Arg182Lys mutation in the helper-component proteinase (HC-ProR182K) that blocks suppression of the miRNA pathway and prevents symptom development in TuGK-infected plants. We exploited TuGK as a viral vector for gain-of-function studies of PHYL1 and SAP54 in Arabidopsis plants. TuGK-PHYL1- and TuGK-SAP54-infected Arabidopsis plants produced identical leafy flower phenotypes and similar gene expression profiles as PHYL1 and SAP54 plants. In addition, the leafy flower formation rate was enhanced in TuGK-PHYL1- or TuGK-SAP54-infected Arabidopsis plants that compared with the T0 lines of PHYL1 plants. These results provide more evidence and novel directions for further studying the mechanism of PHYL1/SAP54-mediated leafy flower development. In addition, the TuGK vector is a good alternative in transgenic plant approaches for rapid gene expression in gain-of-function studies.

The UPR branch IRE1-bZIP60 in plants plays an essential role in viral infection and is complementary to the only UPR pathway in yeast.

The unfolded protein response (UPR) signaling network encompasses two pathways in plants, one mediated by inositol-requiring protein-1 (IRE1)-bZIP60 mRNA and the other by site-1/site-2 proteases (S1P/S2P)-bZIP17/bZIP28. As the major sensor of UPR in eukaryotes, IRE1, in response to endoplasmic reticulum (ER) stress, catalyzes the unconventional splicing of HAC1 in yeast, bZIP60 in plants and XBP1 in metazoans. Recent studies suggest that IRE1p and HAC1 mRNA, the only UPR pathway found in yeast, evolves as a cognate system responsible for the robust UPR induction. However, the functional connectivity of IRE1 and its splicing target in multicellular eukaryotes as well as the degree of conservation of IRE1 downstream signaling effectors across eukaryotes remains to be established. Here, we report that IRE1 and its substrate bZIP60 function as a strictly cognate enzyme-substrate pair to control viral pathogenesis in plants. Moreover, we show that the S1P/S2P-bZIP17/bZIP28 pathway, the other known branch of UPR in plants, does not play a detectable role in virus infection, demonstrating the distinct function of the IRE1-bZIP60 pathway in plants. Furthermore, we provide evidence that bZIP60 and HAC1, products of the enzyme-substrate duet, rather than IRE1, are functionally replaceable to cope with ER stress in yeast. Taken together, we conclude that the downstream signaling of the IRE1-mediated splicing is evolutionarily conserved in yeast and plants, and that the IRE1-bZIP60 UPR pathway not only confers overlapping functions with the other UPR branch in fundamental biology but also may exert a unique role in certain biological processes such as virus-plant interactions.

Effect of biodiversity changes in disease risk: exploring disease emergence in a plant-virus system.

The effect of biodiversity on the ability of parasites to infect their host and cause disease (i.e. disease risk) is a major question in pathology, which is central to understand the emergence of infectious diseases, and to develop strategies for their management. Two hypotheses, which can be considered as extremes of a continuum, relate biodiversity to disease risk: One states that biodiversity is positively correlated with disease risk (Amplification Effect), and the second predicts a negative correlation between biodiversity and disease risk (Dilution Effect). Which of them applies better to different host-parasite systems is still a source of debate, due to limited experimental or empirical data. This is especially the case for viral diseases of plants. To address this subject, we have monitored for three years the prevalence of several viruses, and virus-associated symptoms, in populations of wild pepper (chiltepin) under different levels of human management. For each population, we also measured the habitat species diversity, host plant genetic diversity and host plant density. Results indicate that disease and infection risk increased with the level of human management, which was associated with decreased species diversity and host genetic diversity, and with increased host plant density. Importantly, species diversity of the habitat was the primary predictor of disease risk for wild chiltepin populations. This changed in managed populations where host genetic diversity was the primary predictor. Host density was generally a poorer predictor of disease and infection risk. These results support the dilution effect hypothesis, and underline the relevance of different ecological factors in determining disease/infection risk in host plant populations under different levels of anthropic influence. These results are relevant for managing plant diseases and for establishing conservation policies for endangered plant species.

Differential accumulation of host mRNAs on polyribosomes during obligate pathogen-plant interactions.

Plant pathogens elicit dramatic changes in the expression of host genes during both compatible and incompatible interactions. Gene expression profiling studies of plant-pathogen interactions have only considered messenger RNAs (mRNAs) present in total RNA, which contains subpopulations of actively translated mRNAs associated with polyribosomes (polysomes) and non-translated mRNAs that are not associated with polysomes. The goal of this study was to enhance previous gene expression analyses by identifying host mRNAs that become differentially associated with polysomes following pathogen inoculation. Total and polysomal RNA were extracted from barley (Hordeum vulgare) plants at 32 h after inoculation with Blumeria graminis f. sp. hordei, and Arabidopsis thaliana plants at 10 days after inoculation with Turnip mosaic virus. Gene expression profiles were obtained for each pathosystem, which represent diverse plant host-obligate pathogen interactions. Using this approach, host mRNAs were identified that were differentially associated with polysomes in response to pathogen treatment. Approximately 18% and 26% of mRNAs represented by probe sets on the Affymetrix Barley1 and Arabidopsis ATH1 GeneChips, respectively, differentially accumulated in the two populations in one or more combinations of treatment and genotype. Gene ontology analysis of mRNAs sharing the same pattern of accumulation in total and polysomal RNA identified gene sets that contained a significant number of functionally related annotations, suggesting both transcript accumulation and recruitment to polyribosomes are coordinately regulated in these systems.

A viral deubiquitylating enzyme targets viral RNA-dependent RNA polymerase and affects viral infectivity.

Selective protein degradation via the ubiquitin-proteasome system (UPS) plays an essential role in many major cellular processes, including host-pathogen interactions. We previously reported that the tightly regulated viral RNA-dependent RNA polymerase (RdRp) of the positive-strand RNA virus Turnip yellow mosaic virus (TYMV) is degraded by the UPS in infected cells, a process that affects viral infectivity. Here, we show that the TYMV 98K replication protein can counteract this degradation process thanks to its proteinase domain. In-vitro assays revealed that the recombinant proteinase domain is a functional ovarian tumour (OTU)-like deubiquitylating enzyme (DUB), as is the 98K produced during viral infection. We also demonstrate that 98K mediates in-vivo deubiquitylation of TYMV RdRp protein--its binding partner within replication complexes--leading to its stabilization. Finally, we show that this DUB activity contributes to viral infectivity in plant cells. The identification of viral RdRp as a specific substrate of the viral DUB enzyme thus reveals the intricate interplay between ubiquitylation, deubiquitylation and the interaction between viral proteins in controlling levels of RdRp and viral infectivity.

Arabidopsis DRB4 protein in antiviral defense against Turnip yellow mosaic virus infection.

RNA silencing is an important antiviral mechanism in diverse eukaryotic organisms. In Arabidopsis DICER-LIKE 4 (DCL4) is the primary antiviral Dicer, required for the production of viral small RNAs from positive-strand RNA viruses. Here, we showed that DCL4 and its interacting partner dsRNA-binding protein 4 (DRB4) participate in the antiviral response to Turnip yellow mosaic virus (TYMV), and that both proteins are required for TYMV-derived small RNA production. In addition, our results indicate that DRB4 has a negative effect on viral coat protein accumulation. Upon infection DRB4 expression was induced and DRB4 protein was recruited from the nucleus to the cytoplasm, where replication and translation of viral RNA occur. DRB4 was associated with viral RNA in vivo and directly interacted in vitro with a TYMV RNA translational enhancer, raising the possibility that DRB4 might repress viral RNA translation. In plants the role of RNA silencing in viral RNA degradation is well established, but its potential function in the regulation of viral protein levels has not yet been explored. We observed that severe infection symptoms are not necessarily correlated with enhanced viral RNA levels, but might be caused by elevated accumulation of viral proteins. Our findings suggest that the control of viral protein as well as RNA levels might be important for mounting an efficient antiviral response.

A role for inositol hexakisphosphate in the maintenance of basal resistance to plant pathogens.

Phytic acid (myo-inositol hexakisphosphate, InsP6) is an important phosphate store and signal molecule in plants. However, low-phytate plants are being developed to minimize the negative health effects of dietary InsP6 and pollution caused by undigested InsP6 in animal waste. InsP6 levels were diminished in transgenic potato plants constitutively expressing an antisense gene sequence for myo-inositol 3-phosphate synthase (IPS, catalysing the first step in InsP6 biosynthesis) or Escherichia coli polyphosphate kinase. These plants were less resistant to the avirulent pathogen potato virus Y and the virulent pathogen tobacco mosaic virus (TMV). In Arabidopsis thaliana, mutation of the gene for the enzyme catalysing the final step of InsP6 biosynthesis (InsP5 2-kinase) also diminished InsP6 levels and enhanced susceptibility to TMV and to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae. Arabidopsis thaliana has three IPS genes (AtIPS1-3). Mutant atips2 plants were depleted in InsP6 and were hypersusceptible to TMV, turnip mosaic virus, cucumber mosaic virus and cauliflower mosaic virus as well as to the fungus Botrytis cinerea and to P. syringae. Mutant atips2 and atipk1 plants were as hypersusceptible to infection as plants unable to accumulate salicylic acid (SA) but their increased susceptibility was not due to reduced levels of SA. In contrast, mutant atips1 plants, which were also depleted in InsP6, were not compromised in resistance to pathogens, suggesting that a specific pool of InsP6 regulates defence against phytopathogens.

Proteolytic processing of turnip yellow mosaic virus replication proteins and functional impact on infectivity.

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA.

Genetic and molecular variability of a Turnip mosaic virus population from horseradish (Cochlearia armoracia L.).

Variability and genetic structure of a novel Turnip mosaic virus (TuMV) population from horseradish (Cochlearia armoracia L.) were examined. Over 60 horseradish plants were tested to identify a total of 28 TuMV isolates, constituting the Cochlearia ARmoracia (CAR) TuMV population. Two subgroups of the CAR TuMV isolates could be distinguished: subgroup N did not infect oilseed rape (Brassica napus var. oleifera) cv. Westar plants, while subgroup A infected these plants systemically. Two types of infection of oilseed rape plants were induced by inoculation with the CAR TuMV isolates: systemic mosaic infection and systemic necrotic lesions. The complete sequences of isolates CAR37 (subgroup N) and CAR37A (subgroup A) were determined and compared. The sequences of HC-Pro and CP genes of CAR37 and CAR37A and other isolates of TuMV from other countries were compared to provide some insight into their relatedness. CAR37A, initially regarded as a variant, proved to be very different from CAR37. Re-sequencing after repeated passages confirmed the genetic stability of both isolates.

The poly(A) binding protein is internalized in virus-induced vesicles or redistributed to the nucleolus during turnip mosaic virus infection.

Poly(A) binding protein 2 (PABP2) of Arabidopsis thaliana was previously shown to interact with VPg-Pro of turnip mosaic virus (TuMV) and may consequently play an important role during infection. Subcellular fractionation experiments revealed that PABP2 was predominantly a cytoplasmic soluble protein in healthy plants. However, in TuMV-infected plants, a subpopulation of PABP2 was membrane associated or was localized in the nucleus. Confocal microscopy experiments indicated that PABP2 was partially retargeted to the nucleolus in the presence of TuMV VPg-Pro. In addition, the membrane association of PABP2 during TuMV infection resulted from the internalization of the host protein in 6K-VPg-Pro-induced vesicles, as shown by a combination of confocal microscopy and sucrose gradient fractionation experiments. This redistribution of an important translation initiation factor to the nucleolus and to membrane structure likely underlies two important processes of the TuMV replication cycle.

Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance.

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69(159) and amiR-HC-Pro(159) are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP(159) targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69(159) and amiR-HC-Pro(159) from a dimeric pre-amiR-P69(159)/amiR-HC-Pro(159) transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 degrees C, a temperature that compromises small interfering RNA-mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.

An important determinant of the ability of Turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein.

Turnip mosaic virus (TuMV, genus Potyvirus, family Potyviridae) infects mainly cruciferous plants. Isolates Tu-3 and Tu-2R1 of TuMV exhibit different infection phenotypes in cabbage (Brassica oleracea L.) and Japanese radish (Raphanus sativus L.). Infectious full-length cDNA clones, pTuC and pTuR1, were constructed from isolates Tu-3 and Tu-2R1, respectively. Progeny virus derived from infections with pTuC induced systemic chlorotic and ringspot symptoms in infected cabbage, but no systemic infection in radish. Virus derived from plants infected with pTuR1 induced a mild chlorotic mottle in cabbage and infected radish systemically to induce mosaic symptoms. By exchanging genome fragments between the two virus isolates, the P3-coding region was shown to be responsible for systemic infection by TuMV and the symptoms it induces in cabbage and radish. Moreover, exchanges of smaller parts of the P3 region resulted in recombinants that induced complex infection phenotypes, especially the combination of pTuC-derived N-terminal sequence and pTuR1-derived C-terminal sequence. Analysis by tissue immunoblotting of the inoculated leaves showed that the distributions of P3-chimeric viruses differed from those of the parents, and that the origin of the P3 components affected not only virus accumulation, but also long-distance movement. These results suggest that the P3 protein is an important factor in the infection cycle of TuMV and in determining the host range of this and perhaps other potyviruses.

Targeting of the turnip yellow mosaic virus 66K replication protein to the chloroplast envelope is mediated by the 140K protein.

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two replication proteins, 140K and 66K, both being required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase, and the 66K protein encompasses the RNA-dependent RNA polymerase domain. During viral infection, the 66K protein localizes to virus-induced chloroplastic membrane vesicles, which are closely associated with TYMV RNA replication. To investigate the determinants of its subcellular localization, the 66K protein was expressed in plant protoplasts from separate plasmids. Green fluorescent protein (GFP) fusion and immunofluorescence experiments demonstrated that the 66K protein displayed a cytoplasmic distribution when expressed individually but that it was relocated to the chloroplast periphery under conditions in which viral replication occurred. The 66K protein produced from an expression vector was functional in viral replication since it could transcomplement a defective replication template. Targeting of the 66K protein to the chloroplast envelope in the course of the viral infection appeared to be solely dependent on the expression of the 140K protein. Analysis of the subcellular localization of the 140K protein fused to GFP demonstrated that it is targeted to the chloroplast envelope in the absence of other viral factors and that it induces the clumping of the chloroplasts, one of the typical cytological effects of TYMV infection. These results suggests that the 140K protein is a key organizer of the assembly of the TYMV replication complexes and a major determinant for their chloroplastic localization and retention.

P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction.

The molecular basis for virus-induced disease in plants has been a long-standing mystery. Infection of Arabidopsis by Turnip mosaic virus (TuMV) induces a number of developmental defects in vegetative and reproductive organs. We found that these defects, many of which resemble those in miRNA-deficient dicer-like1 (dcl1) mutants, were due to the TuMV-encoded RNA-silencing suppressor, P1/HC-Pro. Suppression of RNA silencing is a counterdefensive mechanism that enables systemic infection by TuMV. The suppressor interfered with the activity of miR171 (also known as miRNA39), which directs cleavage of several mRNAs coding for Scarecrow-like transcription factors, by inhibiting miR171-guided nucleolytic function. Out of ten other mRNAs that were validated as miRNA-guided cleavage targets, eight accumulated to elevated levels in the presence of P1/HC-Pro. The basis for TuMV- and other virus-induced disease in plants may be explained, at least partly, by interference with miRNA-controlled developmental pathways that share components with the antiviral RNA-silencing pathway.

Stability in vitro of the 69K movement protein of Turnip yellow mosaic virus is regulated by the ubiquitin-mediated proteasome pathway.

Plant viruses move to adjacent cells with the use of virus-encoded cell-to-cell movement proteins. Using proteins produced by in vitro translation, we present evidence that the '69K' movement protein of Turnip yellow mosaic virus (TYMV) is recognized as a substrate for the attachment of polyubiquitin chains and for subsequent rapid and selective proteolysis by the proteasome, the ATP-dependent proteolytic system present in reticulocyte lysate. Truncation of the 69K protein suggests the existence of two degradation signals within its sequence. We propose that selective degradation of virus movement proteins may contribute to the previously reported transient nature of their accumulation during infection.

Mutations in Turnip mosaic virus P3 and cylindrical inclusion proteins are separately required to overcome two Brassica napus resistance genes.

The Brassica napus differential line 165 is resistant to infection by Turnip mosaic virus (TuMV) isolates belonging to pathotypes 1 and 3. Nucleotide sequences of resistance-breaking mutants of pathotype 1 (UK 1), pathotype 3 (CHN 12), and wild-type isolates have been determined. When the mutations identified were introduced into an infectious clone of UK 1, a single mutation in the viral P3 protein induced a hypersensitive (necrotic) response in inoculated leaves of line 165 plants. Full systemic nonnecrotic infection was only possible when another mutation (in the cylindrical inclusion protein) was introduced. Tests on segregating populations derived from line 165 indicated that the two viral genes were pathogenicity determinants for two different resistance genes in line 165. One gene responsible for an extreme form of resistance (no symptoms seen) was epistatic to a second responsible for the hypersensitive reaction. These results help to explain the relative stability of the resistance in line 165 and to further define the genetic basis of the TuMV pathotyping system.

Nickel increases susceptibility of a nickel hyperaccumulator to Turnip mosaic virus.

Hyperaccumulated Ni can defend plant tissues against herbivores and pathogens. The effectiveness of this defense, however, has not been tested with a viral pathogen. Turnip mosaic virus (TuMV) accumulation was studied in two serpentine species of Streptanthus with different Ni uptake abilities. Plants of a Ni hyperaccumulator, milk-wort jewelflower (S. polygaloides Gray), and a non-hyperaccumulator, plumed jewelflower (S. insignis Jepson), were grown on Ni-amended and unamended soils. Plants were inoculated with TuMV at three different phenological stages: basal rosette, bolting, and flowering. Susceptibility of experimental plants to TuMV was determined either by the magnitude of TuMV accumulation (measured by indirect enzyme-linked immunosorbent assay [ELISA]) or by plant survival. Streptanthus polygaloides plants grown on high-Ni soil were more susceptible to TuMV than low-Ni S. polygaloides at all three phenological stages. All rosette and pre-bolt S. insignis plants were infected by TuMV, but survival and TuMV accumulation were not significantly affected by soil Ni. At flowering, only high-Ni S. polygaloides plants became infected. For S. polygaloides, elevated tissue Ni concentrations enhanced TuMV infection instead of defending plants from the virus. To reduce risks to nearby agricultural crops, future phytoremed. iation and phytomining operations using this species should incorporate management plans to prevent the creation of artificial reservoirs of TuMV inoculum.