Genetic diversity and silencing suppression effects of Rice yellow mottle virus and the P1 protein

<p>Abstract</p> <p>Background</p> <p>PTGS (post-transcriptional gene silencing) is used to counter pathogenic invasions, particularly viruses. In return, many plant viruses produce proteins which suppress silencing directed against their RNA. The diversity of silencing suppression at the species level in natural hosts is unknown.</p> <p>Results</p> <p>We investigated the functional diversity of silencing suppression among isolates of the African RYMV (<it>Rice yellow mottle virus</it>) in rice. The RYMV-P1 protein is responsible for cell-to-cell movement and is a silencing suppressor. Transgenic <it>gus</it>-silencing rice lines were used to investigate intra-specific and serogroup silencing suppression diversity at two different levels: that of the virion and the P1 silencing suppressor protein. Our data provide evidence that silencing suppression is a universal phenomenon for RYMV species. However, we found considerable diversity in their ability to suppress silencing which was not linked to RYMV phylogeny, or pathogenicity. At the level of the silencing suppressor P1 alone, we found similar results to those previously found at the virion level. In addition, we showed that cell-to-cell movement of P1 was crucial for the efficiency of silencing suppression. Mutagenesis of P1 demonstrated a strong link between some amino acids and silencing suppression features with, one on the hand, the conserved amino acids C95 and C64 involved in cell-to-cell movement and the strength of suppression, respectively, and on the other hand, the non conserved F88 was involved in the strength of silencing suppression.</p> <p>Conclusion</p> <p>We demonstrated that intra-species diversity of silencing suppression is highly variable and by mutagenesis of P1 we established the first link between silencing suppression and genetic diversity. These results are potentially important for understanding virus-host interactions.</p

Emergence of a resistance breaking isolate of Rice yellow mottle virus during serial inoculations is due to a single substitution in the genome-linked viral protein VPg

International audienceThe recessive gene rymv-1 responsible for the high resistance of Oryza sativa cultivar Gigante to Rice yellow mottle virus (Sobemovirus) was overcome by the variant CI4* which emerged after serial inoculations of the non-resistance-breaking (nRB) isolate CI4. By comparison of the full-length sequences of CI4 and CI4*, we identified a non-synonymous mutation at position 1729 localised in the putative VPg domain and developed an assay based on this single nucleotide polymorphism. The mutation G1729T was detected as early as the first passage in resistant plants and was found in all subsequent passages. Neither reversion nor additional mutation were observed. The substitution G1729T introduced by mutagenesis in the VPg of an nRB infectious clone is sufficient to induce symptoms in non-inoculated leaves of cultivar Gigante. This is the first evidence that VPg is a virulence factor in plants with recessive resistance against viruses outside the family Potyviridae

Rice Yellow Mottle Virus stress responsive genes from susceptible and tolerant rice genotypes

<p>Abstract</p> <p>Background</p> <p>The effects of viral infection involve concomitant plant gene variations and cellular changes. A simple system is required to assess the complexity of host responses to viral infection. The genome of the Rice yellow mottle virus (RYMV) is a single-stranded RNA with a simple organisation. It is the most well-known monocotyledon virus model. Several studies on its biology, structure and phylogeography have provided a suitable background for further genetic studies. 12 rice chromosome sequences are now available and provide strong support for genomic studies, particularly physical mapping and gene identification.</p> <p>Results</p> <p>The present data, obtained through the cDNA-AFLP technique, demonstrate differential responses to RYMV of two different rice cultivars, i.e. susceptible IR64 (<it>Oryza sativa indica</it>), and partially resistant Azucena (<it>O. s. japonica</it>). This RNA profiling provides a new original dataset that will enable us to gain greater insight into the RYMV/rice interaction and the specificity of the host response. Using the SIM4 subroutine, we took the intron/exon structure of the gene into account and mapped 281 RYMV stress responsive (RSR) transcripts on 12 rice chromosomes corresponding to 234 RSR genes. We also mapped previously identified deregulated proteins and genes involved in partial resistance and thus constructed the first global physical map of the RYMV/rice interaction. RSR transcripts on rice chromosomes 4 and 10 were found to be not randomly distributed. Seven genes were identified in the susceptible and partially resistant cultivars, and transcripts were colocalized for these seven genes in both cultivars. During virus infection, many concomitant plant gene expression changes may be associated with host changes caused by the infection process, general stress or defence responses. We noted that some genes (e.g. ABC transporters) were regulated throughout the kinetics of infection and differentiated susceptible and partially resistant hosts.</p> <p>Conclusion</p> <p>We enhanced the first RYMV/rice interaction map by combining information from the present study and previous studies on proteins and ESTs regulated during RYMV infection, thus providing a more comprehensive view on genes related to plant responses. This combined map provides a new tool for exploring molecular mechanisms underlying the RYMV/rice interaction.</p

Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors

[Background] Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis.[Results] Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species.[Conclusion] Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants.P. Baldrich is a recipient of a Ph.D grant from the “Ministerio de Ciencia e Innovación, Formación de Personal Investigador-FPI, ref. BES-2010-032879). This work was supported by grants BIO2009-08719 and BIO2012-32838 to BSS, AGL2010-14949 to JJLM, and BFU2008-04251 to JLR, from the Spanish Ministry of Economy and Competitiveness (MINECO), and grant 2010–0520193 to JLR from the National Science Foundation (NSF). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors

Background: Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis. - Results: Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species. - Conclusion: Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants

Genetic diversity and silencing suppression effects of and the P1 protein-2

Tic delivery (ND), buffer delivery (BD), biolistic delivery with a empty 35S vector (35S), or vectors containing different sP1 from RYMV isolates representative of the viral phylogeny (CI63, Mg1, Tz8, Tz3, BF1). Non transgenic Tai and transgenic L4 served as controls. (A) Photographs correspond to GUS staining at 2 dpd of inoculated leaves. (B) Quantitative effect of different P1 at 2 dpd on -specific siRNA with Northern blot experiments. EtBr staining of rRNA served as a loading control.<p><b>Copyright information:</b></p><p>Taken from "Genetic diversity and silencing suppression effects of and the P1 protein"</p><p>http://www.virologyj.com/content/5/1/55</p><p>Virology Journal 2008;5():55-55.</p><p>Published online 30 Apr 2008</p><p>PMCID:PMC2390521.</p><p></p

Genetic diversity and silencing suppression effects of and the P1 protein-1

Ntrols for -siRNA detection corresponding to RNA from L10 either NI (non-inoculated) or BI (buffer-inoculated) and RNA from non transgenic Tai, or transgenic L4 plants as negative controls. EtBr staining of rRNA served as a loading control. (B) GUS activity is measured by fluorimetry and is expressed as a percentage of the maximal activity measured in CI110 inoculated plants. Analyses were carried out with soluble protein extracted from L10 leaves infected by different isolates. Data represent average values of two independent measurements with standard deviations indicated. (C) Western blot analysis of P1 protein.<p><b>Copyright information:</b></p><p>Taken from "Genetic diversity and silencing suppression effects of and the P1 protein"</p><p>http://www.virologyj.com/content/5/1/55</p><p>Virology Journal 2008;5():55-55.</p><p>Published online 30 Apr 2008</p><p>PMCID:PMC2390521.</p><p></p

Genetic diversity and silencing suppression effects of and the P1 protein-3

Ivery (del) and distal (dis) points after bombardment. (B) Northern analysis of -siRNA and mRNA at 2 days post delivery (dpd) with sTz3, Mg1 and CI63 P1s in the del or dis parts of the leaf. 35S is an empty cassette (negative control) and EtBr staining of rRNA served as a loading control. (C) Western blot analysis of P1 protein. Relative accumulation to the loading control are shown, RNA band intensities were quantified using Image Quant software (Molecular Dynamics).<p><b>Copyright information:</b></p><p>Taken from "Genetic diversity and silencing suppression effects of and the P1 protein"</p><p>http://www.virologyj.com/content/5/1/55</p><p>Virology Journal 2008;5():55-55.</p><p>Published online 30 Apr 2008</p><p>PMCID:PMC2390521.</p><p></p