Amit Gal-On

The Conserved FRNK Box in HC-Pro, a Plant Viral Suppressor of Gene Silencing, Is Required for Small RNA Binding and Mediates Symptom Development

ABSTRACT The helper component-proteinase (HC-Pro) protein of potyviruses is a suppressor of gene silencing and has been shown to elicit plant developmental-defect-like symptoms. In Zucchini yellow mosaic virus (ZYMV), a mutation in the highly conserved FR180NK box of HC-Pro to FI180NK causes attenuation of these symptoms. At 5 days postinoculation and before symptoms appear, virus accumulation, HC-Pro protein levels, and viral short interfering RNA (siRNA) levels are similar for the severe (FRNK) and attenuated (FINK) strains. At this stage, ZYMVFRNK caused greater accumulation of most microRNAs (miRNAs), and especially of their complementary miRNA “passenger” strands (miRNA*s), in systemically infected leaves than the attenuated ZYMVFINK did. HC-ProFRNK specifically bound artificial siRNA and miRNA/miRNA* duplexes with a much higher affinity than the mutated HC-ProFINK. Further analysis of the mutant and wild-type HC-Pro proteins revealed that suppressor activity of the ZYMV HCFINK mutant was not diminished. However, the FINK mutation caused a loss of HC-Pro suppressor function in other potyviruses. Replacement of the second positively charged amino acid in the ZYMV FRNK box to result in FRNA also caused symptom attenuation and reduced small RNA duplex-binding affinity without loss of suppressor activity. Our data suggest that the highly conserved FRNK box in the HC-Pro of potyviruses is a probable point of contact with siRNA and miRNA duplexes. The interaction of the FRNK box with populations of miRNAs directly influences their accumulation levels and regulatory functions, resulting in symptom development.

Characterization of Synergy Between Cucumber mosaic virus and Potyviruses in Cucurbit Hosts

ABSTRACT Mixed infections of cucurbits by Cucumber mosaic virus (CMV) and potyviruses exhibit a synergistic interaction. Zucchini squash and melon plants coinfected by the potyvirus Zucchini yellow mosaic virus (ZYMV) and either Fny-CMV (subgroup IA) or LS-CMV (subgroup II) displayed strong synergistic pathological responses, eventually progressing to vascular wilt and plant death. Accumulation of Fny- or LS-CMV RNAs in a mixed infection with ZYMV in zucchini squash was slightly higher than infection with CMV strains alone. There was an increase in CMV (+) strand RNA levels, but no increase in CMV (-) RNA3 levels during mixed infection with ZYMV. Moreover, only the level of capsid protein from LS-CMV increased in mixed infection. ZYMV accumulated to similar levels in singly and mixed infected zucchini squash and melon plants. Coinfection of squash with the potyvirus Watermelon mosaic virus (WMV) and CMV strains increased both the Fny-CMV RNA levels and the LS-CMV RNA levels. However, CMV (-) strand RNA3 levels were increased little or not at all for CMV on coinfection with WMV. Infection of CMV strains (LS and Fny) containing satellite RNAs (WL47-sat RNA and B5*-sat RNA) reduced the accumulation of the helper virus RNA, except when B5*-sat RNA was mixed with LS- CMV. However, mixed infection containing ZYMV and the CMV strains with satellites reversed the suppression effect of satellite RNAs on helper virus accumulation and increased satellite RNA accumulation. The synergistic interaction between CMV and potyviruses in cucurbits exhibited different features from that documented in tobacco, indicating there are differences in the mechanisms of potyvirus synergistic phenomena.

A Point Mutation in the FRNK Motif of the Potyvirus Helper Component-Protease Gene Alters Symptom Expression in Cucurbits and Elicits Protection Against the Severe Homologous Virus.

Sequence comparison had previously shown three amino acid changes in conserved motifs in the 455-amino acid sequence of the helper component-protease (HC-Pro) between a severe field strain of Zucchini yellow mosaic virus (ZYMV-NAT) and a mild field strain of ZYMV (ZYMV-WK). In this study, exchange of fragments and site-directed mutagenesis within the HC-Pro gene in an infectious clone of ZYMV enabled the effects of the mutations on symptom expression to be mapped. The substitution of Ile for Arg at position 180 in the conserved motif Phe-Arg-Asn-Lys (FRNK) of potyviruses was found to affect symptom expression. Infection of cucurbits with the engineered ZYMV (ZYMV-AG) that contained this mutation caused a dramatic symptom change from severe to mild in squash and to a symptom-free appearance in cucumber, melon, and watermelon. The Ile to Arg mutation was found to be stable, and no revertant virus was found after several passages through plants after long incubation periods. The AG strain was detected 4 days postinoculation and accumulated in cucurbits to a level and with kinetics similar to that of the wild-type ZYMV-AT strain. Cucurbit plants infected with the AG strain were protected against infection by the severe strain.

Gene silencing of mannose 6‐phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant

Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6-phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277-bp fragment from Orobanche aegyptiaca M6PR-mRNA, in an inverted-repeat configuration. M6PR-siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT-PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%-80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene-silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.

Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance.

The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.

Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis

Significance RNAi-mediated antiviral immunity directs specific virus resistance by virus-derived siRNAs in contrast to broad-spectrum resistance triggered in innate immunity by host pattern recognition receptors. Here we show that induction of antiviral RNAi in Arabidopsis is associated with production of a genetically distinct class of virus-activated siRNAs (vasiRNAs) by RNA-dependent RNA polymerase-1 to target hundreds of host genes for RNA silencing by Argonaute-2. Production of vasiRNAs is induced by viruses from two different supergroups of RNA virus families, targeted for inhibition by Cucumber mosaic virus, and correlated with virus resistance independently of viral siRNAs. We propose that antiviral RNAi activates broad-spectrum antiviral activity via widespread silencing of host genes directed by vasiRNAs in addition to specific antiviral defense by viral siRNAs. Antiviral immunity controlled by RNA interference (RNAi) in plants and animals is thought to specifically target only viral RNAs by the virus-derived small interfering RNAs (siRNAs). Here we show that activation of antiviral RNAi in Arabidopsis plants is accompanied by the production of an abundant class of endogenous siRNAs mapped to the exon regions of more than 1,000 host genes and rRNA. These virus-activated siRNAs (vasiRNAs) are predominantly 21 nucleotides long with an approximately equal ratio of sense and antisense strands. Genetically, vasiRNAs are distinct from the known plant endogenous siRNAs characterized to date and instead resemble viral siRNAs by requiring Dicer-like 4 and RNA-dependent RNA polymerase 1 (RDR1) for biogenesis. However, loss of EXORIBONUCLEASE4/THYLENE-INSENSITIVE5 enhances vasiRNA biogenesis and virus resistance without altering the biogenesis of viral siRNAs. We show that vasiRNAs are active in directing widespread silencing of the target host genes and that Argonaute-2 binds to and is essential for the silencing activity of vasiRNAs. Production of vasiRNAs is readily detectable in Arabidopsis after infection by viruses from two distinct supergroups of plant RNA virus families and is targeted for inhibition by the silencing suppressor protein 2b of Cucumber mosaic virus. These findings reveal RDR1 production of Arabidopsis endogenous siRNAs and identify production of vasiRNAs to direct widespread silencing of host genes as a conserved response of plants to infection by diverse viruses. A possible function for vasiRNAs to confer broad-spectrum antiviral activity distinct to the virus-specific antiviral RNAi by viral siRNAs is discussed.

A Nonviral Peptide Can Replace the Entire N Terminus of Zucchini Yellow Mosaic Potyvirus Coat Protein and Permits Viral Systemic Infection

ABSTRACT Systematic deletion and peptide tagging of the amino-terminal domain (NT, ∼43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5′ end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity.

Transgenic resistance to cucumber mosaic virus in tomato: blocking of long-distance movement of the virus in lines harboring a defective viral replicase gene.

ABSTRACT Tomato breeding lines were transformed with a defective replicase gene from RNA 2 of cucumber mosaic virus (CMV). A total of 63 transformants from five tomato genotypes were evaluated for resistance to CMV strains. The responses of R1 transgenic offspring fit into three categories: fully susceptible lines (44%), fully resistant lines (8%), and an intermediate-type mixture of susceptible and resistant seedlings in variable proportions (48%). Further characterization of the response of two highly resistant lines was performed by mechanical inoculation, aphid transmission, or grafting experiments. No virus was detected in noninoculated leaves from these lines, although a low level of virus accumulated initially in the inoculated leaf. The homozygous R2 plants and further generations that were evaluated (up to R5) showed resistance to the Fny-CMV strain, two Israeli isolates tentatively classified as subgroup IA, and K-CMV (a representative of subgroup IB). These lines were partially resistant to LS-CMV (a representative of subgroup II) when a high-virus-titer inoculum was used. Expression of the viral transgene was verified in these lines; however, the expected translation product was not detectable. In grafting experiments, we demonstrated that CMV virions were blocked in their ability to move from infected rootstocks of nontransformed tomato or tobacco into the transgenic scions. Interestingly, virions could not move through a transgenic intersection into the upper scion. These results provide an additional indication that replicase-mediated resistance affects long-distance movement.

Hairpin-based virus resistance depends on the sequence similarity between challenge virus and discrete, highly accumulating siRNA species

Virus resistance can be effectively generated in transgenic plants by using the plant’s silencing machinery. To study the specificity of gene-silencing-based resistance, homozygous tobacco (Nicotiana tabacum L.) plants containing a 597-nt hairpin RNA construct of the Potato Virus Y (PVY) replicase sequence were challenged with a variety of PVY strains. The transgene-carrying tobacco line was immune to five potato PVY strains with high sequence similarity (88.3–99.5%) to the transgene. Infection with more distant tomato and pepper PVY field strains (86–86.8% sequence similarity) caused delayed symptom appearance in the transgenic tobacco. Transgene production of small interfering (si) RNA was detected by northern blot and measured using a custom-designed microarray for the detection of small RNAs. siRNA accumulation peaks were observed throughout the inverted-repeat transgene. In the resistance-breaking tomato and pepper strains there were nucleotide differences in the sequences correlated to siRNA transgene accumulation, indicating the role of siRNA specificity in resistance breaking. The log of transgene siRNA signal intensity increased with probe GC content, indicating that the accumulating siRNA molecules were GC-rich. Sequence similarity of highly accumulating siRNAs with the target virus strain appears to be important for both resistance and resistance-breaking characteristics.

Inoculation of Plants Using Bombardment

This unit describes methods for the construction and use of handheld particle bombardment devices for high-efficiency inoculation of intact plants with nucleic acids and viruses. The devices accelerate heavy metal particles coated with nucleic acids or viruses into plant tissues and are driven by pressurized gas. They are inexpensive to construct and use, and can be assembled in any laboratory. The equipment enables inoculation with full-length infectious cDNA, PCR products, virus from sap or a virus preparation, and in vitro viral transcripts. The inoculation of some phloem-limited RNA viruses is also possible. Additionally, this technology allows for inoculation of large numbers of plants (mass bombardment), inoculation of soft plants that do not survive bombardment inoculation by other means, inoculation in the greenhouse, study of viral recombination in plants, rapid promoter analysis, and monitoring of virus movement using an infectious clone bearing a reporter gene.