Expression of the p24 silencing suppressor of Grapevine leafroll-associated virus 2 from Potato virus X or Barley stripe mosaic virus vector elicits hypersensitive responses in Nicotiana benthamiana.

The 24-kDa protein (p24) encoded by Grapevine leafroll-associated virus 2 (GLRaV-2) is an RNA-silencing suppressor (RSS), but its effect on active viral infection is unclear. Using a Potato virus X (PVX)-based expression system, we demonstrated that p24 elicits lethal systemic necrosis in Nicotiana benthamiana, sharing typical characteristics of the hypersensitive response (HR), and that NbRAR1 (a cytoplasmic Zn2+-binding protein) is involved in the PVX-p24-mediated systemic necrosis. Moreover, expression of p24 from Barley stripe mosaic virus (BSMV) vector triggered local necrosis in infiltrated patches of N. benthamiana, likely inhibiting viral systemic spread. By deletion analysis, we demonstrated that amino acids (aa) 1 to 180, which are located in the region (aa 1-188) previously shown to be necessary for p24's RSS activity, is sufficient for p24 to elicit systemic necrosis in the context of PVX infection. Using substitution mutants, we revealed that silencing-suppression-defective mutants R2A and W54A induce only a mild necrotic response; two mutants without self-interaction ability previously shown to lose or retain weak suppression function also displayed decreased pathogenicity: W149A without RSS activity elicited a mild necrotic response, whereas V162H/L169H/L170H which retains weak RSS activity was able to induce systemic necrosis, but with a 1- to 2-day delay. Taken together, p24 plays an important role in GLRaV-2 pathogenesis, triggering HR-like necrosis in N. benthamiana plants when expressed from PVX or BSMV vector; both the silencing suppression and self-interaction are crucial for p24's pathogenicity activity, and the region of p24 for determining systemic necrosis is mapped to aa 1-180.

Detection of Citrus tristeza virus and Coinfecting Viroids.

Citrus can host a number of important vector- and graft-transmissible pathogens which cause severe diseases. Citrus disease management and clean stock programs require pathogen detection systems which must be economical and sensitive to maintain a healthy citrus industry. Rapid diagnostic tests for simultaneous detection of major graft-transmissible disease agents enable reduction of cost and time. The genetic and biological features of viruses and viroids can vary according to the strains/variants, with severe and mild strains described within the same species. The use of diagnostic tests that can allow to selectively discriminate severe strain(s) is a powerful tool to intercept the most harmful strains and to reduce the need for biological indexing. Moreover a combination of these detection methods will facilitate the studies on the interactions between CTV and viroids, a research topic only partially explored so far.

The p33 protein of Citrus tristeza virus affects viral pathogenicity by modulating a host immune response.

Accumulation of reactive oxygen species (ROS) is a general plant basal defense strategy against viruses. In this study, we show that infection by Citrus tristeza virus (CTV) triggered ROS burst in Nicotiana benthamiana and in the natural citrus host, the extent of which was virus-dose dependent. Using Agrobacterium-mediated expression of CTV-encoded proteins in N. benthamiana, we found that p33, a unique viral protein, contributed to the induction of ROS accumulation and programmed cell death. The role of p33 in CTV pathogenicity was assessed based on gene knockout and complementation in N. benthamiana. In the citrus-CTV pathosystem, deletion of the p33 open reading frame in a CTV variant resulted in a significant decrease in ROS production, compared to that of the wild type CTV, which correlated with invasion of the mutant virus into the immature xylem tracheid cells and abnormal differentiation of the vascular system. By contrast, the wild type CTV exhibited phloem-limited distribution with a minor effect on the vasculature. We conclude that the p33 protein is a CTV effector that negatively affects virus pathogenicity and suggest that N. benthamiana recognizes p33 to activate the host immune response to restrict CTV into the phloem tissue and minimize the disease syndrome.

Characterization of silencing suppressor p24 of Grapevine leafroll-associated virus 2.

Grapevine leafroll-associated virus 2 (GLRaV-2) p24 has been reported to be an RNA silencing suppressor (RSS). However, the mechanisms underlying p24's suppression of RNA silencing are unknown. Using Agrobacterium infiltration-mediated RNA silencing assays, we showed that GLRaV-2 p24 is a strong RSS triggered by positive-sense green fluorescent protein (GFP) RNA, and that silencing suppression by p24 effectively blocks the accumulation of small interfering RNAs. Deletion analyses showed that the region of amino acids 1-188, which contains all predicted α-helices and ß-strands, is required for the RSS activity of p24. Hydrophobic residues I35/F38/V85/V89/W149 and V162/L169/L170, previously shown to be critical for p24 self-interaction, are also crucial for silencing suppression, and western blotting results suggested that a lack of self-interaction ability results in decreased p24 accumulation in plants. The mutants showed greatly weakened or a lack of RSS activity. Substitution with two basic residues at positions 2 or 86, putatively involved in RNA binding, totally abolished the RSS activity of p24, suggesting that p24 uses an RNA-binding strategy to suppress RNA silencing. Our results also showed that W54 in the WG/GW-like motif (W54/G55) is crucial for the RSS activity of p24, whereas p24 does not physically interact with AGO1 of Nicotiana benthamiana. Furthermore, p24 did not promote AGO1 degradation, but significantly up-regulated AGO1 mRNA expression, and this effect was correlated with the RSS activity of p24, indicating that p24 may interfere with microRNA-directed processes. The presented results contribute to our understanding of viral suppression of RNA silencing and the molecular mechanisms underlying GLRaV-2 infection.

Self-interaction of Citrus tristeza virus p33 protein via N-terminal helix.

Citrus tristeza virus (CTV), the most economically important viral pathogen of citrus, encodes a unique protein, p33. CTV p33 shows no similarity with other known proteins, yet plays an important role in viral pathogenesis: it extends the virus host range and mediates virus ability to exclude superinfection by other variants of the virus. Previously we demonstrated that p33 is an integral membrane protein and appears to share characteristics of viral movement proteins. In this study, we show that the p33 protein self-interacts in vitro and in vivo using co-immunoprecipitation, yeast two hybrid, and bimolecular fluorescence complementation assays. Furthermore, a helix located at the N-terminus of the protein is required and sufficient for the protein self-interaction.

Citrus tristeza virus p23: determinants for nucleolar localization and their influence on suppression of RNA silencing and pathogenesis.

Citrus tristeza virus (CTV) encodes a singular protein (p23, 209 amino acids) with multiple functions, including RNA silencing suppression (RSS). Confocal laser-scanning microscopy of green fluorescent protein (GFP)-p23 agroexpressed in Nicotiana benthamiana revealed its accumulation in the nucleolus, Cajal bodies, and plasmodesmata. To dissect the nucleolar localization signal (NoLS) typically associated with basic motifs, seven truncated and 10 point-mutated versions of p23 were assayed. Deletion mutants showed that regions 50 to 86 and 100 to 157 (excluding fragment 106 to 114), both with basic motifs and the first with a zinc-finger, contain the (bipartite) NoLS. Alanine substitutions delimited this signal to three cysteines of the Zn-finger and some basic amino acids. RSS activity of p23 in N. benthamiana was abolished by essentially all mutants, indicating that it involves most p23 regions. The necrotic-inducing ability of p23 when launched in N. benthamiana from Potato virus X was only retained by deletion mutant 158-209 and one substitution mutant, showing that the Zn-finger and flanking basic motifs form part of the pathogenic determinant. Ectopic expression of p23 and some deletion mutants in transgenic Mexican lime demarcated a similar determinant, suggesting that p23 affects related pathways in citrus and N. benthamiana. Both RSS activity and pathogenicity of p23 appear related to its nucleolar localization.

Agroinoculation of Citrus tristeza virus causes systemic infection and symptoms in the presumed nonhost Nicotiana benthamiana.

Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus.

Infection with strains of Citrus tristeza virus does not exclude superinfection by other strains of the virus.

Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were "strains" of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most importantly, build a foundation for the strategy of selecting mild isolates that would efficiently exclude severe virus isolates as a practical means to control CTV diseases.

Tandem leader proteases of Grapevine leafroll-associated virus-2: host-specific functions in the infection cycle.

Several viruses in the genus Closterovirus including Grapevine leafroll-associated virus-2 (GLRaV-2), encode a tandem of papain-like leader proteases (L1 and L2) whose functional profiles remained largely uncharacterized. We generated a series of the full-length, reporter-tagged, clones of GLRaV-2 and demonstrated that they are systemically infectious upon agroinfection of an experimental host plant Nicotiana benthamiana. These clones and corresponding minireplicon derivatives were used to address L1 and L2 functions in GLRaV-2 infection cycle. It was found that the deletion of genome region encoding the entire L1-L2 tandem resulted in a ~100-fold reduction in minireplicon RNA accumulation. Five-fold reduction in RNA level was observed upon deletion of L1 coding region. In contrast, deletion of L2 coding region did not affect RNA accumulation. It was also found that the autocatalytic cleavage by L2 but not by L1 is essential for genome replication. Analysis of the corresponding mutants in the context of N. benthamiana infection launched by the full-length GLRaV-2 clone revealed that L1 or its coding region is essential for virus ability to establish infection, while L2 plays an accessory role in the viral systemic transport. Strikingly, when tagged minireplicon variants were used for the leaf agroinfiltration of the GLRaV-2 natural host, Vitis vinifera, deletion of either L1 or L2 resulted in a dramatic reduction of minireplicon ability to establish infection attesting to a host-specific requirement for tandem proteases in the virus infection cycle.

Diverse suppressors of RNA silencing enhance agroinfection by a viral replicon.

Launching the Beet yellows virus (BYV) minireplicon by agrobacterial delivery resulted in an unexpectedly low number of infected cells per inoculated leaf. This effect was due to a strong RNA silencing response in the agroinfiltrated leaves. Strikingly, ectopic co-expression of p21, a BYV RNA silencing suppressor, increased minireplicon infectivity by three orders of magnitude. Mutational analysis demonstrated that this effect correlates with suppressor activity of p21. Five diverse, heterologous viral suppressors were also active in this system, providing a useful approach for a dramatic, up to 10,000-fold, increase of the efficiency of agroinfection. The minireplicon agroinfection assay was also used to identify a new suppressor, a homolog of BYV p21, derived from Grapevine leafroll-associated virus-2. In addition, we report preliminary data on the suppressor activity of the p10 protein of Grapevine virus A and show that this protein belongs to a family of Zn-ribbon-containing proteins encoded by filamentous plant RNA viruses from three genera. The members of this family are predicted to have RNA silencing suppressor activity.

Viral-like symptoms induced by the ectopic expression of the p23 gene of Citrus tristeza virus are citrus specific and do not correlate with the pathogenicity of the virus strain.

Ectopic expression of the p23 gene from a severe (T36) strain of Citrus tristeza virus (CTV) induces viral-like symptoms in Mexican lime. Here, we report that expressing the same gene from a mild strain induced similar symptoms that correlated with accumulation of p23 protein irrespective of the source strain. CTV inoculation of transgenic limes showing CTV-like leaf symptoms and high p23 accumulation did not modify symptoms initially, with the virus titer being as in inoculated nontransgenic controls; however, at later stages, symptoms became attenuated. Transformation with p23-T36 of CTV-susceptible sweet and sour orange and CTV-resistant trifoliate orange also led to CTV-like leaf symptoms that did not develop when plants were transformed with a truncated p23 version. In transgenic citrus species and relatives other than Mexican lime, p23 was barely detectable, although symptom intensity correlated with levels of p23 transcripts. The lower accumulation of p23 in sweet and sour orange compared with Mexican lime also was observed in nontransgenic plants inoculated with CTV, suggesting that minimal p23 levels cause deleterious effects in the first two species. Conversely, transgenic expression of p23 in CTV nonhost Nicotiana spp. led to accumulation of p23 without phenotypic aberrations, indicating that p23 interferes with plant development only in citrus species and relatives.

Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome.

Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the approximately 20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F(1) plants expressing p23 and not from the CP- or p20-expressing F(1) plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host.

Detection of beet yellows closterovirus methyltransferase-like and helicase-like proteins in vivo using monoclonal antibodies.

In the positive-stranded RNA genome of beet yellows closterovirus (BYV), the 5'-terminal ORF 1a encodes a 295 kDa polyprotein with the domains of papain-like cysteine proteinase, methyltransferase (MT) and helicase (HEL), whereas ORF 1b encodes an RNA-dependent RNA polymerase. Eleven and five hybridoma cell lines secreting monoclonal antibodies (MAbs) were derived from mice injected with the bacterially expressed fragments of the BYV 1a product encompassing the MT and HEL domains, respectively. On immunoblots of protein from BYV-infected Tetragonia expansa plants, four MAbs against the MT recognized a approximately 63 kDa protein, and two MAbs against the HEL recognized a approximately 100 kDa protein. Both the methyltransferase-like protein and the helicase-like protein were found mainly in the fractions of large organelles (P1) and membranes (P30) of the infected plants. These data clearly indicate that (i) the BYV methyltransferase-like and helicase-like proteins, like other related viral enzymes, are associated with membrane compartments in cells, and (ii) the 1a protein, apart from the cleavage by the leader papain-like proteinase that is expected to produce the 66 kDa and 229 kDa fragments, undergoes additional processing by a virus-encoded or cellular proteinase.

In vitro transcripts from cloned cDNAs of the lettuce infectious yellows closterovirus bipartite genomic RNAs are competent for replication in Nicotiana benthamiana protoplasts.

Full-length cloned cDNAs of lettuce infectious yellows closterovirus (LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter. To assess RNA replication, Nicotiana benthamiana protoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derived in vitro transcripts. Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG) in vitro transcripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions. Furthermore, protoplasts inoculated with only capped in vitro transcripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgRNA, indicating that LIYV RNA 1 can replicate in the absence of LIYV RNA 2. Conversely, accumulation of LIYV RNA 2 was not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-derived capped in vitro transcripts. These data demonstrate that LIYV genomic RNAs are competent for replication in mesophyll protoplasts and that infectious in vitro transcripts can be derived from the cloned cDNAs of a closterovirus genome.