Structure-function analyses of coiled-coil immune receptors define a hydrophobic module for improving plant virus resistance.

Plant immunity relies on nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) that detect microbial patterns released by pathogens, and activate localized cell death to prevent the spread of pathogens. Tsw is the only identified resistance (R) gene encoding an NLR, conferring resistance to tomato spotted wilt orthotospovirus (TSWV) in pepper species (Capsicum, Solanaceae). However, molecular and cellular mechanisms of Tsw-mediated resistance are still elusive. Here, we analysed the structural and cellular functional features of Tsw protein, and defined a hydrophobic module to improve NLR-mediated virus resistance. The plasma membrane associated N-terminal 137 amino acid in the coiled-coil (CC) domain of Tsw is the minimum fragment sufficient to trigger cell death in Nicotiana benthamiana plants. Transient and transgenic expression assays in plants indicated that the amino acids of the hydrophobic groove (134th-137th amino acid) in the CC domain is critical for its full function and can be modified for enhanced disease resistance. Based on the structural features of Tsw, a super-hydrophobic funnel-like mutant, TswY137W, was identified to confer higher resistance to TSWV in a SGT1 (Suppressor of G-two allele of Skp1)-dependent manner. The same point mutation in a tomato Tsw-like NLR protein also improved resistance to pathogens, suggesting a feasible way of structure-assisted improvement of NLRs.

Red-light is an environmental effector for mutualism between begomovirus and its vector whitefly.

Environments such as light condition influence the spread of infectious diseases by affecting insect vector behavior. However, whether and how light affects the host defense which further affects insect preference and performance, remains unclear, nor has been demonstrated how pathogens co-adapt light condition to facilitate vector transmission. We previously showed that begomoviral ßC1 inhibits MYC2-mediated jasmonate signaling to establish plant-dependent mutualism with its insect vector. Here we show red-light as an environmental catalyzer to promote mutualism of whitefly-begomovirus by stabilizing ßC1, which interacts with PHYTOCHROME-INTERACTING FACTORS (PIFs) transcription factors. PIFs positively control plant defenses against whitefly by directly binding to the promoter of terpene synthase genes and promoting their transcription. Moreover, PIFs interact with MYC2 to integrate light and jasmonate signaling and regulate the transcription of terpene synthase genes. However, begomovirus encoded ßC1 inhibits PIFs' and MYC2' transcriptional activity via disturbing their dimerization, thereby impairing plant defenses against whitefly-transmitted begomoviruses. Our results thus describe how a viral pathogen hijacks host external and internal signaling to enhance the mutualistic relationship with its insect vector.

Functional analysis of the HS185 regulatory element in the rice HSP70 promoter.

A series of HSP70 promoter deletion constructs was established. Analysis of beta-glucuronidase activities from the promoter deletion constructs in transient expression assays identified a cis-element, located from -493 to -308 bp upstream of the ATG start site. This element was designated as HS185 and has a crucial role in HSP70 promoter activity. HS185 has some characteristics of a miniature inverted-repeat transposable element (MITE), such as terminal inverted repeats (TIRs) (GGTCCCACA) and a putative target site duplication. There are 362 copies of homologous sequences of HS185 in the rice genome, which are preferentially distributed to non-coding regions. Based on these sequence features, we propose that HS185 is an uncharacterized rice MITE, possibly derived from the rice transposon Mutator-like element VIII family. Further transient expression assays showed that HS185 inhibited the enhancer activity of the cauliflower mosaic virus 35S promoter. These results demonstrate that not only is HS185 necessary for HSP70 promoter activity, but it also has a functional role as an insulator. This study explored new regulatory functions of non-coding repeat sequences in rice.

Satellite RNA-derived small interfering RNA satsiR-12 targeting the 3' untranslated region of Cucumber mosaic virus triggers viral RNAs for degradation.

RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. The most frequently cloned satsiRNA, satsiR-12, was predicted to imperfectly match to Cucumber mosaic virus (CMV) RNAs in the upstream area of the 3' untranslated region (3' UTR). Moreover, an artificial satsiR-12 (asatsiR-12) mediated cleavage of a green fluorescent protein (GFP) sensor construct harboring the satsiR-12 target site. asatsiR-12 also mediated reduction of viral RNAs in 2b-deficient CMV (CMVΔ2b)-infected Nicotiana benthamiana. The reduction was not observed in CMVΔ2b-infected RDR6i plants, in which RDR6 was silenced. Following infection with 2b-containing CMV, the reduction in viral RNAs was not observed in plants of either genotype, indicating that the asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b protein. Our results suggest that satsiR-12 targeting the 3' UTR of CMV RNAs triggered RDR6-dependent antiviral silencing. Competition experiments with wild-type CMV RNAs and anti-satsiR-12 mutant RNA1 in the presence of 2b and satRNA demonstrate the inhibitory effect of the 2b protein on the satsiR-12-related degradation of CMV RNAs, revealing a substantial suppressor function of the 2b protein in native CMV infection. Our data provide evidence for the important biological functions of satsiRNAs in homeostatic interactions among the host, virus, and satRNA in the final outcome of viral infection.

Satellite RNA reduces expression of the 2b suppressor protein resulting in the attenuation of symptoms caused by Cucumber mosaic virus infection.

Satellite RNAs (satRNAs) depend on cognate helper viruses for replication, encapsidation, movement and transmission. Many satRNAs with different symptom modulation effects have been reported. The pathogenicity of satRNAs is thought to be the result of a direct interaction among the satRNA, helper viruses and host factors by unknown mechanisms. To understand the effect of satRNA of Cucumber mosaic virus (a severe field ShanDong strain, SD-CMV) on pathogenicity, and the possible involvement of host RNA silencing pathways in pathogenicity, we constructed biologically active CMV cDNA clones and a CMV-Δ2b mutant lacking the open reading frame of 2b, a silencing suppressor protein, in order to infect Nicotiana benthamiana and Arabidopsis with or without SD-satRNA. We found that SD-satRNA reduced the accumulation of the 2b protein and its coding RNA4A and attenuated the yellowing caused by SD-CMV infection. Small RNA analysis indicated that the 2b protein interfered with RNA silencing, specifically in the synthesis of CMV RNA3-derived small interfering RNAs (R3-siRNAs). The accumulation of R3-siRNAs in CMV-Δ2b infection was reduced in the presence of satRNA, for which greater accumulation of satRNA-derived siRNAs (satsiRNAs) was detected. Our results suggest that abundant SD-satRNA serving as target for RNA silencing may play a role in protecting helper CMV RNA, especially, subgenomic RNA4, from being targeted by RNA silencing. This compensates for the increase in RNA silencing resulting from the reduction in expression of the 2b suppressor in the presence of satRNA. Our data provide evidence that a plant silencing mechanism is involved in the pathogenicity of satRNA.

RNA-dependent RNA polymerase 1 from Nicotiana tabacum suppresses RNA silencing and enhances viral infection in Nicotiana benthamiana.

Endogenous eukaryotic RNA-dependent RNA polymerases (RDRs) produce double-stranded RNA intermediates in diverse processes of small RNA synthesis in RNA silencing pathways. RDR6 is required in plants for posttranscriptional gene silencing induced by sense transgenes (S-PTGS) and has an important role in amplification of antiviral silencing. Whereas RDR1 is also involved in antiviral defense in plants, this does not necessarily proceed through triggering silencing. In this study, we show that Nicotiana benthamiana transformed with RDR1 from Nicotiana tabacum (Nt-RDR1 plants) exhibits hypersusceptibility to Plum pox potyvirus and other viruses, resembling RDR6-silenced (RDR6i) N. benthamiana. Analysis of transient induction of RNA silencing in N. benthamiana Nt-RDR1 and RDR6i plants revealed that Nt-RDR1 possesses silencing suppression activity. We found that Nt-RDR1 does not interfere with RDR6-dependent siRNA accumulation but turns out to suppress RDR6-dependent S-PTGS. Our results, together with previously published data, suggest that RDR1 might have a dual role, contributing, on one hand, to salicylic acid-mediated antiviral defense, and suppressing, on the other hand, the RDR6-mediated antiviral RNA silencing. We propose a scenario in which the natural loss-of-function variant of RDR1 in N. benthamiana may be the outcome of selective pressure to maintain a high RDR6-dependent antiviral defense, which would be required to face the hypersensitivity of this plant to a large number of viruses.

A critical domain of the Cucumber mosaic virus 2b protein for RNA silencing suppressor activity.

Alignment of Cucumber mosaic virus (CMV) 2b protein sequences from two CMV subgroups revealed two highly variable regions. To examine contributions of variable sequence domains to the suppressor activity, we performed a comparative study between 2b proteins of a subgroup I strain (SD-CMV) and a subgroup II strain (Q-CMV). Here we show that the suppressor activity of SD2b is stronger than that of Q2b and that a domain existent in SD2b but absent in Q2b is a major determinant of the suppressor activity of SD2b. We further show that the same domain is responsible for inhibition of Nicotiana benthamiana AGO4-1 transcription. Our results implicate AGO4 as a mediator for CMV 2b to suppress systemic silencing and DNA methylation.

Identification of a movement protein of rice yellow stunt rhabdovirus.

Rice yellow stunt rhabdovirus (RYSV) encodes seven genes in its negative-sense RNA genome in the order 3'-N-P-3-M-G-6-L-5'. The existence of gene 3 in the RYSV genome and an analogous gene(s) of other plant rhabdoviruses positioned between the P and M genes constitutes a unique feature for plant rhabdoviruses that is distinct from animal-infecting rhabdoviruses in which the P and M genes are directly linked. However, little is known about the function of these extra plant rhabdovirus genes. Here we provide evidence showing that the protein product encoded by gene 3 of RYSV, P3, possesses several properties related to a viral cell-to-cell movement protein (MP). Analyses of the primary and secondary protein structures suggested that RYSV P3 is a member of the "30K" superfamily of viral MPs. Biolistic bombardment transcomplementation experiments demonstrated that RYSV P3 can support the intercellular movement of a movement-deficient potexvirus mutant in Nicotiana benthamiana leaves. In addition, Northwestern blot analysis indicated that the RYSV P3 protein can bind single-stranded RNA in vitro, a common feature of viral MPs. Finally, glutathione S- transferase pull-down assays revealed a specific interaction between the RYSV P3 protein and the N protein which is a main component of the ribonucleocapsid, a subviral structure believed to be involved in the intercellular movement of plant rhabdoviruses. Together, these data suggest that RYSV P3 is likely a MP of RYSV, thus representing the first example of characterized MPs for plant rhabdoviruses.

[Using green fluorescent protein as a reporter to monitor elimination of selectable marker genes from transgenic plants].

In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.

Novel structure of the genome of Rice yellow stunt virus: identification of the gene 6-encoded virion protein.

The genomic region encompassing the L protein gene and a small open reading frame (ORF 6) of Rice yellow stunt virus (RYSV) has been sequenced, thus completing the nucleotide sequence of the RYSV genome. The genome organization of RYSV is unique in the rhabdoviruses because it contains two additional genes when compared to the basic gene order of the family Rhabdoviridae: Phylogenetic analysis revealed that the amino acid sequence of the RYSV L protein is most closely related to that of the L protein of Sonchus yellows net virus, another nucleorhabdovirus. However, the RYSV L protein has a unique acidic N-terminal domain distinct from that of other rhabdoviruses. Moreover, the polypeptide encoded by the ORF 6 was detected by immunoblot analysis in purified RYSV virions. Thus RYSV provides the first example in the family Rhabdoviridae that a small ORF between the G and L genes encodes a virion protein.

The promoter of a rice glycine-rich protein gene, Osgrp-2, confers vascular-specific expression in transgenic plants.

The genomic sequence of a rice (Oryza sativa L.) glycine-rich protein (GRP) gene, designated Osgrp-2, has been previously determined (GenBank U40708). Primer extension analysis indicated that transcription starts 47 bp upstream of the translation start codon. To gain an insight into the transcriptional regulation of this gene, the 2,401-bp promoter sequence and a series of its 5' deletions were transcriptionally fused to the beta-glucuronidase (GUS) gene. GUS activity was subsequently assayed in a transient expression system of tobacco ( Nicotiana tabacum L.) protoplasts, which revealed the presence of a positive regulatory region (-2290 to -1406) and two negative regulatory regions (-2401 to -2291 and -1405 to -1022) in the Osgrp-2 promoter for the promoter activity. The positive regulatory region displayed an enhancer-like activity when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter (-89 to +6) to drive GUS expression and assayed on tobacco leaves by the Agrobacterium-mediated transient expression technique (agroinfiltration). Histochemical staining for GUS activity on transgenic tobacco plants has further indicated a preferential expression in vascular tissues of stems and leaves conferred by the positive regulatory region. A 1,023-bp fragment of the Osgrp-2 promoter (-1021 to +2) fused with GUS was transformed into tobacco and proved to be capable of conferring vascular-specific expression. Further 5' and 3' deletion analysis of the 1,023-bp promoter revealed that a 99-bp fragment located from -497 to -399 contained cis-elements responsible for vascular-specific expression.