Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles.

Autophagy plays a paramount role in mammalian antiviral immunity including direct targeting of viruses and their individual components, and many viruses have evolved measures to antagonize or even exploit autophagy mechanisms for the benefit of infection. In plants, however, the functions of autophagy in host immunity and viral pathogenesis are poorly understood. In this study, we have identified both anti- and proviral roles of autophagy in the compatible interaction of cauliflower mosaic virus (CaMV), a double-stranded DNA pararetrovirus, with the model plant Arabidopsis thaliana We show that the autophagy cargo receptor NEIGHBOR OF BRCA1 (NBR1) targets nonassembled and virus particle-forming capsid proteins to mediate their autophagy-dependent degradation, thereby restricting the establishment of CaMV infection. Intriguingly, the CaMV-induced virus factory inclusions seem to protect against autophagic destruction by sequestering capsid proteins and coordinating particle assembly and storage. In addition, we found that virus-triggered autophagy prevents extensive senescence and tissue death of infected plants in a largely NBR1-independent manner. This survival function significantly extends the timespan of virus production, thereby increasing the chances for virus particle acquisition by aphid vectors and CaMV transmission. Together, our results provide evidence for the integration of selective autophagy into plant immunity against viruses and reveal potential viral strategies to evade and adapt autophagic processes for successful pathogenesis.

Complete nucleotide sequence of strawberry vein banding virus Chinese isolate and infectivity of its full-length DNA clone.

BACKGROUND: Strawberry vein banding virus (SVBV) is a double-stranded DNA plant virus, which has been found in North America, Australia, Brazil, Japan, Europe and several provinces of China. Infected strawberry plants exhibit mild vein-banding symptoms and chlorosis along the veins. It is one of the most economically important diseases in Asiatic, European and North American strawberry-growing areas. FINDINGS: The complete genome of an SVBV Chinese isolate (SVBV-CN) was isolated and cloned from a naturally infected strawberry (Fragaria × ananassa cv. Sachinoka) sample found in Shenyang city of Liaoning province. Sequence analysis revealed a complete genome of 7864 nucleotides (nts) that indicated SVBV-CN was most closely related to SVBV from the United States (SVBV-US) with a sequence similarity of 85.8 %. Two major clades were identified based on phylogenetic analysis of the complete genome sequences of caulimoviruses. SVBV-CN clustered together with SVBV-US, whereas other caulimoviruses formed a separate branch. Agrobacterium-mediated inoculation of Fragaria vesca with an infectious clone of SVBV-CN results in systemic infection with distinct symptoms of yellowing bands along the main leaf veins. This suggests that the SVBV-CN infectious clone can recapitulate the symptoms observed in naturally infected strawberries, and therefore is likely the causal agent of the original disease observed in strawberries. Furthermore, strawberry plants inoculated with the infectious clone using vacuum infiltration developed symptoms with a very high infection rate of 86-100 % in 4-5 weeks post-inoculation. This compares to an infection rate of 20-40 % in 8-9 weeks post-inoculation using syringe-inoculation. CONCLUSIONS: The complete nucleotide sequence of SVBV from a naturally infected strawberry was determined. Agroinfiltration of strawberry plants using an infectious clone of SVBV-CN resulted in symptoms typically found in infected strawberries from Shenyang city of Liaoning province in China. This is the first report describing an infectious clone of SVBV-CN, and that vacuum infiltration can be potentially used as a new and highly efficient means for inoculation of strawberry plants.

Reduction of leaf area and symptom severity as proxies of disease-induced plant mortality: the example of the Cauliflower mosaic virus infecting two Brassicaceae hosts.

Disease induced effects on host survival are important to understand the evolution of parasitic virulence and host resistance/tolerance. Unfortunately, experiments evaluating such effects are in most cases logistically demanding justifying the measurement of survival proxies. For plant hosts commonly used proxies are leaf area and the nature and severity of visual qualitative disease symptoms. In this study we tested whether these traits are indeed correlated to the host mortality rate induced by viral infection. We infected Brassica rapa and Arabidopsis thaliana plants with different natural isolates of Cauliflower mosaic virus (CaMV) and estimated over time the development of symptoms and the relative reduction of leaf area compared to healthy plants and followed plant mortality. We observed that the mortality of infected plants was correlated with the relative reduction of leaf area of both B. rapa and A. thaliana. Measures of mortality were also correlated with the severity of visual qualitative symptoms but the magnitude of the correlations and the time frame at which they were significant depended on the host plant: stronger and earlier correlations were observed on A. thaliana.

Antiviral activity of tenofovir against Cauliflower mosaic virus and its metabolism in Brassica pekinensis plants.

The antiviral effect of the acyclic nucleoside phosphonate tenofovir (R)-PMPA on double-stranded DNA Cauliflower mosaic virus (CaMV) in Brassica pekinensis plants grown in vitro on liquid medium was evaluated. Double antibody sandwich ELISA and PCR were used for relative quantification of viral protein and detecting nucleic acid in plants. (R)-PMPA at concentrations of 25 and 50 mg/l significantly reduced CaMV titers in plants within 6-9 weeks to levels detectable neither by ELISA nor by PCR. Virus-free plants were obtained after 3-month cultivation of meristem tips on semisolid medium containing 50 mg/l (R)-PMPA and their regeneration to whole plants in the greenhouse. Studying the metabolism of (R)-PMPA in B. pekinensis revealed that mono- and diphosphate, structural analogs of NDP and/or NTP, are the only metabolites formed. The data indicate very low substrate activity of the enzymes toward (R)-PMPA as substrate. The extent of phosphorylation in the plant's leaves represents only 4.5% of applied labeled (R)-PMPA. In roots, we detected no radioactive peaks of phosphorylated metabolites of (R)-PMPAp or (R)-PMPApp.

Application of game theory to the interaction between plant viruses during mixed infections.

Natural mixed infections of plant viruses are frequent, often leading to unpredictable variations in symptoms, infectivity, accumulation and/or vector transmissibility. Cauliflower mosaic caulimovirus (CaMV) has often been found in mixed infections with turnip mosaic potyvirus (TuMV) in plants of the genus Brassica. This study addressed the effect of mixed infection on infectivity, pathogenicity and accumulation of CaMV and TuMV in Arabidopsis thaliana plants inoculated mechanically with cDNA infectious clones. In singly infected plants, TuMV accumulation was approximately 8-fold higher than that of CaMV. In co-infected plants, there was 77 % more TuMV accumulation compared with single infections, whilst the accumulation of CaMV was 56 % lower. This outcome describes a biological game in which TuMV always plays the winner strategy, leading to the competitive exclusion of CaMV. However, the infectivity of each virus was not affected by the presence of the other, and no symptom synergism was observed.

Enhanced resistance and neutralization of defense responses by suppressors of RNA silencing.

The effects of transgenic expression of the potato virus Y (PVY) HCPro silencing suppressor in tobacco were examined on infection by several viruses. Infection by tobacco mosaic virus (TMV) was reduced at 25 degrees C, but not at 33 degrees C. By contrast, systemic infection at 33 degrees C by the TMV expressing green fluorescent protein was promoted by the HCPro. Infection by tobacco rattle virus (TRV) was restricted to local necrotic lesions by the PVY HCPro. However, this resistance was neutralized by expression of the cucumber mosaic virus (CMV) 2b protein from TRV. By contrast, infection by either wild-type CMV or CMV with a deletion of the 2b gene was not affected. Similarly, infection by cauliflower mosaic virus, red clover necrotic mosaic virus (both limited to infection of the inoculated leaves of tobacco) or tomato bushy stunt virus (systemically infecting tobacco) was not altered by the expression of PVY HCPro. Therefore, it appeared that the PVY HCPro was able to induce defense responses at 25 degrees C, but not at 33 degrees C, where it actually neutralized a pre-existing defense response. Moreover, the CMV 2b protein was able to neutralize a defense response activated by HCPro in combination with TRV.

Self-interaction of ORF II protein through the leucine zipper is essential for Soybean chlorotic mottle virus infectivity.

The ORF II protein (PII) of Soybean chlorotic mottle virus (SbCMV) is essential for the virus life cycle. We investigated the interactions of SbCMV PII with itself and with other essential virus proteins using a Gal4-based yeast two-hybrid system. PII interacted only with itself and not with any other virus proteins. The PII-PII interaction was confirmed by a Sos-based yeast two-hybrid system and a far-western analysis. Deletion mutagenesis mapped the self-interacting domain to the C-terminal 48 amino acids (amino acids 154-201), which contain two putative leucine zipper motifs. Introduction of amino acid substitutions to leucine/isoleucine in zipper sequences prevented the PII-PII interaction and abolished the infectivity of SbCMV. These results revealed that the self-interaction of PII through a leucine zipper is necessary for virus infection.

Arabidopsis mutants that suppress the phenotype induced by transgene-mediated expression of cauliflower mosaic virus (CaMV) gene VI are less susceptible to CaMV-infection and show reduced ethylene sensitivity.

Protein P6 is the main symptom determinant of cauliflower mosaic virus (CaMV), and transgene-mediated expression in Arabidopsis induces a symptom-like phenotype in the absence of infection. Seeds of a P6-transgenic line, A7, were mutagenized by gamma-irradiation and M2 seedlings were screened for mutants that suppressed the phenotype of chlorosis and stunting. We identified four mutants that were larger and less chlorotic than the A7 parent but which contained an intact and transcriptionally active transgene. The two mutants with the strongest suppression phenotype, were recessive and allelic. The transgene was eliminated by back-crossing with wild-type Arabidopsis. In progeny lines that were homozygous for the putative suppressor mutation the proportion of plants becoming infected following inoculation with CaMV was 40% that of wild-type, although in plants that did become infected, levels of virus DNA in mutants and wild-type did not differ significantly. Symptoms in the mutants were milder and delayed although this was somewhat dependent on the virus isolate. This phenotype was inherited stably. Both mutant alleles showed a partially ethylene-insensitive phenotype in an ethylene triple response assay. P6-transgenic plants were also almost completely insensitive to ethylene in the triple response assay. We suggest that the chlorosis and stunting in P6-transgenic and CaMV-infected plants are dependent on interactions between P6 and components involved in ethylene signalling, and that the suppressor gene product may function to augment these interactions.

Activation of three pathogen-inducible promoters of tobacco in transgenic pear (Pyrus communis L.) after abiotic and biotic elicitation.

In order to improve pear resistance against fire blight caused by Erwinia amylovora, a search for promoters driving high-level expression of transgenes specifically in response to this bacterial pathogen has been undertaken. We have examined the ability of hsr203J, str246C and sgd24 tobacco (Nicotiana tabacum L.) promoters to drive expression of the uidA reporter gene in pear. Transgenic pear clones were obtained by Agrobacterium tumefaciens-mediated transformation. Beta-glucuronidase activity was determined quantitatively and qualitatively in these plants grown in vitro using fluorometric and histochemical assays and compared to cauliflower mosaic virus (CaMV) 35S promoter-driven activity. The hsr203J promoter appeared to be very weakly activated following inoculation in pear, which is the converse of the situation in tobacco. The str246C promoter was rapidly activated in pear during compatible and incompatible interactions, by wounding and following the application of several elicitors (capsicein, cryptogein, harpin, salicylic acid and jasmonic acid). The sgd24 promoter, a deletion derivative of str246C, exhibited a low level of expression after bacterial inoculation, was weakly activated by wounding and elicitors, and was not activated by phytohormones (salicylic acid and jasmonic acid). Interestingly, the sgd24 promoter was locally activated in pear, whereas the str246C promoter was activated systemically from the infection site. Taken together, these data show that, although the s tr246C and sgd24 promoters are less active than the CaMV35S promoter in pear, their pathogen-responsiveness would permit them to be used to drive the expression of transgenes to promote bacterial disease resistance.

Differential induction of symptoms in Arabidopsis by P6 of Cauliflower mosaic virus.

The gene VI protein (P6) of Cauliflower mosaic virus (CaMV) functions as a virulence factor in crucifers by eliciting chlorotic symptoms in infected plants. The ability to induce chlorosis has been associated previously with P6 through gene-swapping experiments between strains and through the development of transgenic plants that express P6. The primary role that has been identified for P6 in the CaMV infection cycle is to modify the host translation machinery to facilitate the translation of the polycistronic CaMV 35S RNA. This function for P6 has been designated as the translational transactivator (TAV) function. In the present study, we have characterized an unusual variant of P6, derived from CaMV strain D4, that does not induce chlorosis upon transformation into Arabidopsis thaliana. The level of D4 P6 produced in transgenic Arabidopsis line D4-2 was comparable to the amount found in transgenic plants homozygous for W260 and CM1841 P6, two versions of P6 that induce strong chlorotic symptoms and stunting in Arabidopsis. A complementation assay proved that P6 expressed in the D4-2 line was functional, as it could support the systemic infection of a CM1841 mutant that contained a lethal frame-shift mutation within gene VI. This complementation assay allowed us to separately assess the contribution of CM1841 gene VI to symptom development versus the contribution of other CM1841 genes. Furthermore, a previous study had shown that the TAV activity of D4 P6 was comparable to that of W260 P6. That comparative analysis of TAV function, coupled with the characterization of the D4-2 transgenic line in the present paper, indicates that the TAV function of P6 may play only a minor role in the development of chlorotic symptoms.

Multiple domains within the Cauliflower mosaic virus gene VI product interact with the full-length protein.

The Cauliflower mosaic virus (CaMV) gene VI product (P6) is a multifunctional protein essential for viral propagation. It is likely that at least some of these functions require P6 self-association. The work described here was performed to confirm that P6 self-associates and to identify domains involved in this interaction. Yeast two-hybrid analyses indicated that full-length P6 self-associates and that this interaction is specific. Additional analyses indicated that at least four independent domains bind to full-length P6. When a central domain (termed domain D3) was removed, these interactions were abolished. However, this deleted P6 was able to bind to the full-length wild-type protein and to isolated domain D3. Viruses lacking domain D3 were incapable of producing a systemic infection. Isolated domain D3 was capable of binding to at least two of the other domains but was unable to self-associate. This suggests that domain D3 facilitates P6 self-association by binding to the other domains but not itself. The presence of multiple domains involved in P6 self-association may help explain the ability of this protein to form the intracellular inclusions characteristic of caulimoviruses.

Risk assessment of virus-resistant transgenic plants.

Virus-resistant transgenic plants (VRTPs) hold the promise of enormous benefit for agriculture. However, over the past ten years, questions concerning the potential ecological impact of VRTPs have been raised. In some cases, detailed study of the mode of action of the resistance gene has made it possible to eliminate the source of potential risk, notably the possible effects of heterologous encapsidation on the transmission of viruses by their vectors. In other cases, the means of eliminating likely sources of risk have not yet been developed. When such residual risk still exists, the potential risks associated with the VRTP must be compared with those associated with nontransgenic plants so that risk assessment can fully play its role as part of an overall analysis of the advantages and disadvantages of practicable solutions to the problem solved by the VRTP.

Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms.

A new allele of the coronatine-insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1-20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1-20 plants is correlated with hyperactivation of PR-1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA-mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1-20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA-mediated defenses are required for restriction of PstDC3000 growth in coi1-20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1-20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1-20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA-dependent defense pathway; and (ii) an SA-independent inability to develop disease symptoms. These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.

Genes Ia, II, III, IV and V of Soybean chlorotic mottle virus are essential but the gene Ib product is non-essential for systemic infection.

Soybean chlorotic mottle virus (SbCMV) is the type species of the genus 'Soybean chlorotic mottle-like viruses', within the family Caulimoviridae. The double-stranded DNA genome of SbCMV (8178 bp) contains eight major open reading frames (ORFs). Viral genes essential and non-essential for the replication and movement of SbCMV were investigated by mutational analysis of an infectious 1.3-mer DNA clone. The results indicated that ORFs Ia, II, III, IV and V were essential for systemic infection. The product of ORF Ib was non-essential, although the putative tRNA(Met) primer-binding site in ORF Ib was proved to be essential. Immunoselection PCR revealed that an ORF Ia deletion mutant was encapsidated in primarily infected cells, indicating that ORF Ia is required for virus movement but not for replication. ORF IV was confirmed to encode a capsid protein by peptide sequencing of the capsid. Analysis of the viral transcripts showed that the SbCMV DNA genome gives rise to a pregenomic RNA and an ORF VI mRNA, as shown in the case of Cauliflower mosaic virus.

The promoter of a Brassica napus lipid transfer protein gene is active in a range of tissues and stimulated by light and viral infection in transgenic Arabidopsis.

cDNA and genomic clones encoding Brassica napus non-specific lipid transfer proteins (LTP) were isolated and sequenced. The encoded amino acid sequences were very similar to those reported previously for LTPs from B. napus and other species. Sequence information indicates that B. napus contains an LTP gene family. The 5'-flanking region of one gene, designated BnLTP, was fused to GUS and the fusion introduced into Arabidopsis. LTP transcripts and BnLTP-Gus expression were present predominantly in the epidermis of leaf and stem, consistent with the hypothesised function of LTPs in the deposition of cuticular or epicuticular waxes. However, GUS activity was detected in other tissues, including lateral root initials, anthers, stigmas and vascular tissues, which may suggest additional functions. LTP transcript levels in B. napus and Arabidopsis and BnLTP-GUS expression in transgenic Arabidopsis were stimulated by blue and red light but not UV-B. BnLTP promoter activity was also stimulated upon viral infection, at a time when the virus had spread systemically. No increase in expression was observed in response to cold or wounding.

Requirement of cauliflower mosaic virus open reading frame VI product for viral gene expression and multiplication in turnip protoplasts.

Cauliflower mosaic virus (CaMV) open reading frame (ORF) VI product (P6) has been shown to be the major constituent of viral inclusion body, to function as a post-transcriptional transactivator, and to be essential for infectivity on whole plants. Although these findings suggest that P6 has an important role in viral multiplication, it is unknown whether P6 is required for viral multiplication in a single cell. To address this question, we transfected turnip protoplasts with an ORF VI frame-shift (4 bp deletion) mutant (pCaFS6) of an infectious CaMV DNA clone (pCa122). The mutant was uninfectious. Co-transfection of plasmids expressing P6 complemented the mutant. Overexpression of P6 elevated the infection rate in co-transfection experiments with either pCa122 or pCaFS6. This would have been achieved by elevating the level of pregenomic 35S RNA, a putative polycistronic mRNA for ORFs I, II, III, IV and V, and by enhancing the accumulation of these five viral gene products. When CaMV ORFs I, II, III, IV and V were expressed from monocistronic constructs in which each of the ORFs was placed just downstream of the 35S promoter, the accumulation of ORF III, IV and V products depended on the co-expression of P6. The accumulation of ORF I and II products was not detected, even in the presence of P6. These results suggest that P6 is involved in the stabilization of other viral gene products as well as in the activation of viral gene expression, and thus, is a prerequisite for CaMV multiplication.

Alternative structures of the cauliflower mosaic virus 35 S RNA leader: implications for viral expression and replication.

The CaMV 35 S RNA functions as both messenger and pregenomic RNA under the control of its 600 nts leader, which contains regulatory elements involved in splicing, polyadenylation, translation, reverse transcription, and probably also packaging. The structure of the leader has been characterized theoretically and experimentally. The predicted conformation, a low-energy elongated hairpin, base-pairing the two halves of the leader, with a cross-like structure at the top, is strongly supported by enzymatic probing, chemical modification, and phylogenetic comparison. The elongated hairpin is stabilized by strong base-pairing between the ends of the leader, regions which are important in allowing translation downstream of the leader via the ribosome shunt mechanism. At high ionic strength the 35 S RNA leader exhibits additional higher order structures of low electrophoretic mobility: (1) a long-range pseudoknot connecting central and terminal parts of the leader; (2) a dimer. Alternative structures of the CaMV 35 S RNA leader may co-exist and have specialized functions. Their potential impact on CaMV life cycle regulation is discussed.

Identification of structural domains within the cauliflower mosaic virus movement protein by scanning deletion mutagenesis and epitope tagging.

Plant viruses encode proteins that mediate their movement from cell to cell through plasmodesmata. Currently, the mechanisms of action of these movement proteins (MPs) can be divided broadly into two types, requiring or not requiring the presence of viral capsid protein. Cauliflower mosaic virus encodes a multifunctional MP (P1) that modifies plasmodesmata through the formation of tubules that contain virus particles. To investigate the structure of P1, 26 small deletions (scanning deletions) were used to characterize regions of P1 essential for full biological activity. These deletions identified an N-terminal region and a region close to but not at the C terminus as domains that could tolerate manipulation, although gross deletions of either domain abolished infection. In sequence comparisons with other caulimovirus MPs, these regions coincided with the areas of least amino acid homology. Epitope tags inserted into either of these regions were stably maintained in systemic infections, and in extracts from infected plants, tagged P1 was detected on immunoblots. We predicted that, from the hypervariability of these regions, they would be located on the surface of the native P1 structure. Immunofluorescence of P1-specific tubules formed on the surface of infected protoplasts confirmed that the N-terminal and C terminus-proximal regions were exposed on the surface of the P1 tubule subunit. These findings establish a structure for P1 that is likely to be applicable to other tubule-forming MPs.