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.

Identification of a novel microRNA (miRNA) from rice that targets an alternatively spliced transcript of the Nramp6 (Natural resistance-associated macrophage protein 6) gene involved in pathogen resistance.

Plants have evolved efficient defence mechanisms to defend themselves from pathogen attack. Although many studies have focused on the transcriptional regulation of defence responses, less is known about the involvement of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in plant immunity. This work investigates miRNAs that are regulated by elicitors from the blast fungus Magnaporthe oryzae in rice (Oryza sativa). Small RNA libraries were constructed from rice tissues and subjected to high-throughput sequencing for the identification of elicitor-responsive miRNAs. Target gene expression was examined by microarray analysis. Transgenic lines were used for the analysis of miRNA functioning in disease resistance. Elicitor treatment is accompanied by dynamic alterations in the expression of a significant number of miRNAs, including new members of annotated miRNAs. Novel miRNAs from rice are proposed. We report a new rice miRNA, osa-miR7695, which negatively regulates an alternatively spliced transcript of OsNramp6 (Natural resistance-associated macrophage protein 6). This novel miRNA experienced natural and domestication selection events during evolution, and its overexpression in rice confers pathogen resistance. This study highlights an miRNA-mediated regulation of OsNramp6 in disease resistance, whilst illustrating the existence of a novel regulatory network that integrates miRNA function and mRNA processing in plant immunity.

An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves.

BACKGROUND: Localized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNAi signal. In cereals, however, the morphology of the leaf and particularly the structure of the leaf epidermis, prevent infiltration of a bacterial suspension in cells by simple pressure, a method otherwise successful in dicots leaves. This study aimed at establishing a rapid method for the functional analysis of rice genes based on the triggering of RNA interference (RNAi) following Agrobacterium-mediated transient transformation of leaves. RESULTS: Using an agro-infection protocol combining a wound treatment and a surfactant, we were able to obtain in a reliable manner transient expression of a T-DNA-borne uidA gene in leaf cells of japonica and indica rice cultivars. Using this protocol to transiently inhibit gene expression in leaf cells, we introduced hairpin RNA (hpRNA) T-DNA constructs containing gene specific tags of the phytoene desaturase (OsPDS) and of the SLENDER 1 (OsSLR1) genes previously proven to trigger RNAi of target genes in stable transformants. SiRNA accumulation was observed in the agro-infected leaf area for both constructs indicating successful triggering of the silencing signal. Accumulation of secondary siRNA was observed in both stably and transiently transformed leaf tissues expressing the HpRNA OsSLR1 construct. Gene silencing signalling was investigated in monitoring the parallel time course of OsPDS-derived mRNA and siRNA accumulation in the agro-infiltrated leaf area and adjacent systemic sectors. The sensitive RT-Q-PCR method evidenced a consistent, parallel decrease of OsPDS transcripts in both the agroinfiltred and adjacent tissues, with a time lag for the latter. CONCLUSIONS: These results indicate that the method is efficient at inducing gene silencing in the agro-infected leaf area. The transfer of low amounts of siRNA, probably occurring passively through the symplastic pathway from the agro-infected area, seemed sufficient to trigger degradation of target transcripts in the adjacent tissues. This method is therefore well suited to study the cell-to-cell movement of the silencing signal in a monocot plant and further test the functionality of natural and artificial miRNA expression constructs.

Diurnal oscillation in the accumulation of Arabidopsis microRNAs, miR167, miR168, miR171 and miR398.

MicroRNAs (miRNAs) are small RNAs acting as regulators of eukaryotic gene expression at the post-transcriptional level. Plant miRNAs have been implicated in developmental processes and adaptation to the environment. We show that the accumulation of four Arabidopsis miRNAs (miR171, miR398, miR168 and miR167) oscillates during the diurnal cycle, their accumulation increasing during the light period of the daytime and decreasing in darkness. This oscillatory pattern of miRNA accumulation is not governed by the circadian clock. These results suggest a potential role of light in controlling miRNA accumulation while defining a new level of regulation of miRNA gene expression in Arabidopsis.

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

BACKGROUND: 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. RESULTS: We investigated the functional diversity of silencing suppression among isolates of the African RYMV (Rice yellow mottle virus) in rice. The RYMV-P1 protein is responsible for cell-to-cell movement and is a silencing suppressor. Transgenic gus-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. CONCLUSION: 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.

Rice yellow mottle virus stress responsive genes from susceptible and tolerant rice genotypes.

BACKGROUND: 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. RESULTS: The present data, obtained through the cDNA-AFLP technique, demonstrate differential responses to RYMV of two different rice cultivars, i.e. susceptible IR64 (Oryza sativa indica), and partially resistant Azucena (O. s. japonica). 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. CONCLUSION: 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.

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.

The recessive gene rymv-1, responsible for the high resistance of Oryza sativa 'Gigante' to Rice yellow mottle virus (genus 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*, a non-synonymous mutation was identified at position 1729, localized in the putative VPg domain, and an assay was developed 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 any additional mutation was observed. The substitution G1729T, introduced by mutagenesis into the VPg of an nRB infectious clone, was sufficient to induce symptoms in uninoculated leaves of O. sativa 'Gigante'. This is the first evidence that VPg is a virulence factor in plants with recessive resistance against viruses outside the family Potyviridae.