XA21-mediated resistance to Xanthomonas oryzae pv. oryzae is dose dependent.

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21. These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.

OsMKK1 is a novel element that positively regulates the Xa21-mediated resistance response to Xanthomonas oryzae pv. oryzae in rice.

KEY MESSAGE: OsMKK1, a MAPK gene, positively regulates rice Xa21-mediated resistance response and also plays roles in normal growth and development process of rice. The mitogen-activated protein kinase (MAPK) cascade was highly conserved among eukaryotes, which played crucial roles in plant responses to pathogen infection. Bacterial blight is the most devastating bacterial disease. Xa21 confers broad-spectrum resistance to Xanthomonas oryzae pv. Oryzae (Xoo). This study identified that the transcription level of OsMKK1 was up-regulated in resistant response against Xoo, thus overexpression (OsMKK1-OX) and RNA interference (OsMKK1-RNAi) transgenic rice lines under the background of Xa21 was constructed. Compared with recipient control plants 4021, the OsMKK1-OX lines significantly enhanced disease resistance to Xoo, on the contrary, the resistance of OsMKK1-RNAi lines was weakened, demonstrated that OsMKK1 played a positive role in Xa21-mediated disease resistance pathway. A number of pathogenesis-related proteins, including PR1A, PR2 and PR10A showed enhanced expression in OsMKK1-OX lines, supported that these PR genes may be regulated by OsMKK1 to participate in the defense responses. In addition, the agronomic traits of OsMKK1 transgenic plants were affected. Overall, these results revealed the role of OsMKK1 in Xa21-mediated resistance against Xoo and in the normal growth and development process in rice.

SnRK1A-mediated phosphorylation of a cytosolic ATPase positively regulates rice innate immunity and is inhibited by Ustilaginoidea virens effector SCRE1.

Rice false smut caused by Ustilaginoidea virens is becoming one of the most recalcitrant rice diseases worldwide. However, the molecular mechanisms underlying rice immunity against U. virens remain unknown. Using genetic, biochemical and disease resistance assays, we demonstrated that the xb24 knockout lines generated in non-Xa21 rice background exhibit an enhanced susceptibility to the fungal pathogens U. virens and Magnaporthe oryzae. Consistently, flg22- and chitin-induced oxidative burst and expression of pathogenesis-related genes in the xb24 knockout lines were greatly attenuated. As a central mediator of energy signaling, SnRK1A interacts with and phosphorylates XB24 at Thr83 residue to promote ATPase activity. SnRK1A is activated by pathogen-associated molecular patterns and positively regulates plant immune responses and disease resistance. Furthermore, the virulence effector SCRE1 in U. virens targets host ATPase XB24. The interaction inhibits ATPase activity of XB24 by blocking ATP binding to XB24. Meanwhile, SCRE1 outcompetes SnRK1A for XB24 binding, and thereby suppresses SnRK1A-mediated phosphorylation and ATPase activity of XB24. Our results indicate that the conserved SnRK1A-XB24 module in multiple crop plants positively contributes to plant immunity and uncover an unidentified molecular strategy to promote infection in U. virens and a novel host target in fungal pathogenesis.

Rice MPK17 Plays a Negative Role in the Xa21-Mediated Resistance Against Xanthomonas oryzae pv. oryzae.

Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases affecting rice production worldwide. Xa21 was the first disease resistance gene cloned in rice, which encodes a receptor kinase and confers broad resistance against Xoo stains. Dozens of components in the Xa21-mediated pathway have been identified in the past decades, however, the involvement of mitogen-activated protein kinase (MAPK) genes in the pathway has not been well described. To identify MAPK involved in Xa21-mediated resistance, the level of MAPK proteins was profiled using Western blot analysis. The abundance of OsMPK17 (MPK17) was found decreased during the rice-Xoo interaction in the background of Xa21. To investigate the function of MPK17, MPK17-RNAi and over-expression (OX) transgenic lines were generated. The RNAi lines showed an enhanced resistance, while OX lines had impaired resistance against Xoo, indicating that MPK17 plays negative role in Xa21-mediated resistance. Furthermore, the abundance of transcription factor WRKY62 and pathogenesis-related proteins PR1A were changed in the MPK17 transgenic lines when inoculated with Xoo. We also observed that the MPK17-RNAi and -OX rice plants showed altered agronomic traits, indicating that MPK17 also plays roles in the growth and development. On the basis of the current study and published results, we propose a "Xa21-MPK17-WRKY62-PR1A" signaling that functions in the Xa21-mediated disease resistance pathway. The identification of MPK17 advances our understanding of the mechanism underlying Xa21-mediated immunity, specifically in the mid- and late-stages.

Silencing of Dicer-like protein 2a restores the resistance phenotype in the rice mutant, sxi4 (suppressor of Xa21-mediated immunity 4).

The rice immune receptor XA21 confers resistance to Xanthomonas oryzae pv. oryzae (Xoo), and upon recognition of the RaxX21-sY peptide produced by Xoo, XA21 activates the plant immune response. Here we screened 21 000 mutant plants expressing XA21 to identify components involved in this response, and reported here the identification of a rice mutant, sxi4, which is susceptible to Xoo. The sxi4 mutant carries a 32-kb translocation from chromosome 3 onto chromosome 7 and displays an elevated level of DCL2a transcript, encoding a Dicer-like protein. Silencing of DCL2a in the sxi4 genetic background restores resistance to Xoo. RaxX21-sY peptide-treated leaves of sxi4 retain the hallmarks of XA21-mediated immune response. However, WRKY45-1, a known negative regulator of rice resistance to Xoo, is induced in the sxi4 mutant in response to RaxX21-sY peptide treatment. A CRISPR knockout of a short interfering RNA (TE-siRNA815) in the intron of WRKY45-1 restores the resistance phenotype in sxi4. These results suggest a model where DCL2a accumulation negatively regulates XA21-mediated immunity by altering the processing of TE-siRNA815.

Plant immunity: Rice XA21-mediated resistance to bacterial infection.

In this article, we describe the development of the plant immunity field, starting with efforts to understand the genetic basis for disease resistance, which ∼30 y ago led to the discovery of diverse classes of immune receptors that recognize and respond to infectious microbes. We focus on knowledge gained from studies of the rice XA21 immune receptor that recognizes RaxX (required for activation of XA21 mediated immunity X), a sulfated microbial peptide secreted by the gram-negative bacterium Xanthomonas oryzae pv. oryzae. XA21 is representative of a large class of plant and animal immune receptors that recognize and respond to conserved microbial molecules. We highlight the complexity of this large class of receptors in plants, discuss a possible role for RaxX in Xanthomonas biology, and draw attention to the important role of sulfotyrosine in mediating receptor-ligand interactions.

The HrpX Protein Activates Synthesis of the RaxX Sulfopeptide, Required for Activation of XA21-Mediated Immunity to Xanthomonas oryzae pv. oryzae.

Upon encountering a susceptible plant host, a bacterial pathogen expresses specific virulence factors. For example, in planta, the Xanthomonas HrpX protein activates transcription of roughly 150 genes encoding components of the type III secretion system or its translocated effectors, as well as other secreted proteins implicated in pathogenesis. Here, we show that X. oryzae pv. oryzae growth in planta or in HrpX-inducing XOM2 media resulted in HrpX-dependent transcription of the raxX and raxST genes that control production of the RaxX sulfopeptide, exported through a type I secretion system. The RaxX protein is required for activation of XA21-mediated immunity in Xa21+ rice lines. We identified potential plant-inducible promoter elements upstream of the likely 5' ends of the raxX and raxST transcripts. Deletions and nucleotide substitutions confirmed that these elements are required for HrpX-dependent expression of raxX and raxST. We conclude that raxX-raxST gene expression is induced by HrpX during growth in planta and, therefore, is coordinately expressed with other genes required for pathogenesis.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Paladin, a tyrosine phosphatase-like protein, is required for XA21-mediated immunity in rice.

XA21 encodes a rice immune receptor that confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo). XA21-mediated immunity is triggered by recognition of a small protein called RaxX-sY (required for activation of XA21-mediated immunity X, tyrosine-sulfated) secreted by Xoo. To identify components regulating XA21-mediated immunity, we generated and screened a mutant population of fast-neutron-mutagenized rice expressing Ubi:Myc-XA21 for those susceptible to Xoo. Here, we report the characterization of one of these rice mutants, named sxi2 (suppressor of XA21-mediated immunity-2). Whole-genome sequencing revealed that sxi2 carries a deletion of the PALADIN (PALD) gene encoding a protein with three putative protein tyrosine phosphatase-like domains (PTP-A, -B, and -C). Expression of PALD in the sxi2 genetic background was sufficient to complement the susceptible phenotype, which requires the catalytic cysteine of the PTP-A active site to restore resistance. PALD co-immunoprecipitated with the full-length XA21 protein, whose levels are positively regulated by the presence of the PALD transgene. Furthermore, we foundd that sxi2 retains many hallmarks of XA21-mediated immunity, similar to the wild type. These results reveal that PALD, a previously uncharacterized class of phosphatase, functions in rice innate immunity, and suggest that the conserved cysteine in the PTP-A domain of PALD is required for its immune function.

The Rice Xa3 Gene Confers Resistance to Xanthomonas oryzae pv. oryzae in the Model Rice Kitaake Genetic Background.

The rice XA21 and XA3 pattern receptor kinases, derived from Oryza longistaminata and an Oryza. sativa japonica cultivar Wase Aikoku 3, respectively, confer resistance to strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. Previously, we showed that transfer of Xa21 to the model rice cultivar Kitaake enhances resistance to Xoo. In this manuscript we demonstrate that Kitaake expressing Xa3 confers resistance to Xoo strain PXO79 and that the stress-related marker genes PR10b and KO5 are upregulated in Xoo-infected Xa3 rice leaves. We also show that rice somatic embryogenesis receptor kinase 2 (OsSERK2) positively regulates XA3-mediated immunity in Kitaake. We found that overexpression of XA21 binding protein 15 (XB15) and XB24, two negative regulators of XA21-mediated immunity, do not affect XA3-mediated immunity in the Kitaake genetic background. Our results indicate that the rice immune receptors XA21 and XA3 employ both shared and distinct signaling components in their response to Xoo. The results are important to further understand pathogen-associated molecular pattern (PAMP)-triggered immunity in rice. Furthermore, the presence of Kitaake rice carrying Xa3 will facilitate genetic research to study the XA3-mediated immunity.

Genome sequence of the model rice variety KitaakeX.

BACKGROUND: The availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to transform and propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species. RESULTS: Here, we report the de novo genome sequencing and analysis of Oryza sativa ssp. japonica variety KitaakeX, a Kitaake plant carrying the rice XA21 immune receptor. Our KitaakeX sequence assembly contains 377.6 Mb, consisting of 33 scaffolds (476 contigs) with a contig N50 of 1.4 Mb. Complementing the assembly are detailed gene annotations of 35,594 protein coding genes. We identified 331,335 genomic variations between KitaakeX and Nipponbare (ssp. japonica), and 2,785,991 variations between KitaakeX and Zhenshan97 (ssp. indica). We also compared Kitaake resequencing reads to the KitaakeX assembly and identified 219 small variations. The high-quality genome of the model rice plant KitaakeX will accelerate rice functional genomics. CONCLUSIONS: The high quality, de novo assembly of the KitaakeX genome will serve as a useful reference genome for rice and will accelerate functional genomics studies of rice and other species.

Application of high-throughput amplicon sequencing-based SSR genotyping in genetic background screening.

BACKGROUND: Host genetic backgrounds affect gene functions. The genetic backgrounds of genetically engineered organisms must be identified to confirm their genetic backgrounds identity with those of recipients. Marker-assisted backcrossing (MAB), transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) editing are three commonly used genetic engineering techniques. However, methods for genetic background screening between genetically engineered organisms and corresponding recipients suffer from low efficiency, low accuracy or high cost. RESULTS: Here, we improved our previously reported AmpSeq-SSR method, an amplicon sequencing-based simple sequence repeat (SSR) genotyping method, by selecting SSR loci with high polymorphism among varieties. Ultimately, a set of 396 SSRs was generated and applied to evaluate the genetic backgrounds identity between rice lines developed through MAB, transgenesis, and CRISPR/Cas9 editing and the respective recipient rice. We discovered that the percentage of different SSRs between the MAB-developed rice line and its recipient was as high as 23.5%. In contrast, only 0.8% of SSRs were different between the CRISPR/Cas9-system-mediated rice line and its recipient, while no SSRs showed different genotypes between the transgenic rice line and its recipient. Furthermore, most differential SSRs induced by MAB technology were located in non-coding regions (62.9%), followed by untranslated regions (21.0%) and coding regions (16.1%). Trinucleotide repeats were the most prevalent type of altered SSR. Most importantly, all altered SSRs located in coding regions were trinucleotide repeats. CONCLUSIONS: This method is not only useful for the background evaluation of genetic resources but also expands our understanding of the unintended effects of different genetic engineering techniques. While the work we present focused on rice, this method can be readily extended to other organisms.

Variation and inheritance of the Xanthomonas raxX-raxSTAB gene cluster required for activation of XA21-mediated immunity.

The rice XA21-mediated immune response is activated on recognition of the RaxX peptide produced by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The 60-residue RaxX precursor is post-translationally modified to form a sulfated tyrosine peptide that shares sequence and functional similarity with the plant sulfated tyrosine (PSY) peptide hormones. The 5-kb raxX-raxSTAB gene cluster of Xoo encodes RaxX, the RaxST tyrosylprotein sulfotransferase, and the RaxA and RaxB components of a predicted type I secretion system. To assess raxX-raxSTAB gene cluster evolution and to determine its phylogenetic distribution, we first identified rax gene homologues in other genomes. We detected the complete raxX-raxSTAB gene cluster only in Xanthomonas spp., in five distinct lineages in addition to X. oryzae. The phylogenetic distribution of the raxX-raxSTAB gene cluster is consistent with the occurrence of multiple lateral (horizontal) gene transfer events during Xanthomonas speciation. RaxX natural variants contain a restricted set of missense substitutions, as expected if selection acts to maintain peptide hormone-like function. Indeed, eight RaxX variants tested all failed to activate the XA21-mediated immune response, yet retained peptide hormone activity. Together, these observations support the hypothesis that the XA21 receptor evolved specifically to recognize Xoo RaxX.

Four tyrosine residues of the rice immune receptor XA21 are not required for interaction with the co-receptor OsSERK2 or resistance to Xanthomonas oryzae pv. oryzae.

Tyrosine phosphorylation has emerged as an important regulator of plasma membrane-localized immune receptors activity. Here, we investigate the role of tyrosine phosphorylation in the regulation of rice XANTHOMONAS RESISTANCE 21 (XA21)-mediated immunity. We demonstrate that the juxtamembrane and kinase domain of Escherichia coli-expressed XA21 (XA21JK) autophosphorylates on tyrosine residues. Directed mutagenesis of four out of the nine tyrosine residues in XA21JK reduced autophosphorylation. These sites include Tyr698 in the juxtamembrane domain, and Tyr786, Tyr907, and Tyr909 in the kinase domain. Rice plants expressing XA21-GFP fusion proteins or proteins with these tyrosine residues individually mutated to phenylalanine (XA21YF-GFP), which prevents phosphorylation at these sites, maintain resistance to Xanthomonas oryzae pv. oryzae. In contrast, plants expressing phosphomimetic XA21 variants with tyrosine mutated to aspartate (XA21YD-GFP) were susceptible. In vitro purified XA21JKY698F, XA21JKY907F, and XA21JKY909F variants are catalytically active, whereas activity was not detected in XA21JKY768F and the four XA21JKYD variants. We previously demonstrated that interaction of XA21 with the co-receptor OsSERK2 is critical for biological function. Four of the XA21JKYF variants maintain interaction with OsSERK2 as well as the XA21 binding (XB) proteins XB3 and XB15 in yeast, suggesting that these four tyrosine residues are not required for their interaction. Taken together, these results suggest that XA21 is capable of tyrosine autophosphorylation, but the identified tyrosine residues are not required for activation of XA21-mediated immunity or interaction with predicted XA21 signaling proteins.

RaxM regulates the AvrXa21 (RaxX)-mediated immune response.

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease, which causes a reduction in rice production. The interaction between Xoo and rice is a model for the study of the gene-for-gene hypothesis, in which a resistance (R) gene encoding a product interacts with an effector molecule encoded by a corresponding bacterial avirulence (avr) gene. Rice XA21 functions as a plant innate immune receptor (R protein) and recognizes the avirulence protein (RaxX) of Xoo to induce the immune response and cope with pathogen attack. The sulphuration of RaxX by the tyrosine sulphotransferase RaxST is essential to its activity. The expression of raxST is regulated by the RaxH/RaxR and phoP/phoQ two-component systems. However, the regulation of raxX expression remains unclear. Here, we showed that a gene (raxM) encodes a small protein, which functions as a regulator of raxX expression. raxX and raxM are located upstream of raxST. Transcriptional analysis indicates that raxX and raxM are separately transcribed and the promoter of raxX is located at the raxM coding region. The RaxM protein regulates its own and raxX expression, and is required for the XA21-mediated immunity response. Therefore, we identified a regulator of raxX expression and of the Xoo-rice interaction. Our findings suggest that RaxX is not only regulated at the post-translational level, but also at the transcriptional level.

The rice XA21 ectodomain fused to the Arabidopsis EFR cytoplasmic domain confers resistance to Xanthomonas oryzae pv. oryzae.

Rice (Oryza sativa) plants expressing the XA21 cell-surface receptor kinase are resistant to Xanthomonas oryzae pv. oryzae (Xoo) infection. We previously demonstrated that expressing a chimeric protein containing the ELONGATION FACTOR Tu RECEPTOR (EFR) ectodomain and the XA21 endodomain (EFR:XA21) in rice does not confer robust resistance to Xoo. To test if the XA21 ectodomain is required for Xoo resistance, we produced transgenic rice lines expressing a chimeric protein consisting of the XA21 ectodomain and EFR endodomain (XA21:EFR) and inoculated these lines with Xoo. We also tested if the XA21:EFR rice plants respond to a synthetic sulfated 21 amino acid derivative (RaxX21-sY) of the activator of XA21-mediated immunity, RaxX. We found that five independently transformed XA21:EFR rice lines displayed resistance to Xoo as measured by lesion length analysis, and showed that five lines share characteristic markers of the XA21 defense response (generation of reactive oxygen species and defense response gene expression) after treatment with RaxX21-sY. Our results indicate that expression of the XA21:EFR chimeric receptor in rice confers resistance to Xoo. These results suggest that the endodomain of the EFR and XA21 immune receptors are interchangeable and the XA21 ectodomain is the key determinant conferring robust resistance to Xoo.

Molecular mimicry modulates plant host responses to pathogens.

Background: Pathogens often secrete molecules that mimic those present in the plant host. Recent studies indicate that some of these molecules mimic plant hormones required for development and immunity. Scope and Conclusion: This Viewpoint reviews the literature on microbial molecules produced by plant pathogens that functionally mimic molecules present in the plant host. This article includes examples from nematodes, bacteria and fungi with emphasis on RaxX, a microbial protein produced by the bacterial pathogen Xanthomonas oryzae pv. oryzae. RaxX mimics a plant peptide hormone, PSY (plant peptide containing sulphated tyrosine). The rice immune receptor XA21 detects sulphated RaxX but not the endogenous peptide PSY. Studies of the RaxX/XA21 system have provided insight into both host and pathogen biology and offered a framework for future work directed at understanding how XA21 and the PSY receptor(s) can be differentially activated by RaxX and endogenous PSY peptides.

Overexpression of Rice Auxilin-Like Protein, XB21, Induces Necrotic Lesions, up-Regulates Endocytosis-Related Genes, and Confers Enhanced Resistance to Xanthomonas oryzae pv. oryzae.

BACKGROUND: The rice immune receptor XA21 confers resistance to the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). To elucidate the mechanism of XA21-mediated immunity, we previously performed a yeast two-hybrid screening for XA21 interactors and identified XA21 binding protein 21 (XB21). RESULTS: Here, we report that XB21 is an auxilin-like protein predicted to function in clathrin-mediated endocytosis. We demonstrate an XA21/XB21 in vivo interaction using co-immunoprecipitation in rice. Overexpression of XB21 in rice variety Kitaake and a Kitaake transgenic line expressing XA21 confers a necrotic lesion phenotype and enhances resistance to Xoo. RNA sequencing reveals that XB21 overexpression results in the differential expression of 8735 genes (4939 genes up- and 3846 genes down-regulated) (≥2-folds, FDR ≤0.01). The up-regulated genes include those predicted to be involved in 'cell death' and 'vesicle-mediated transport'. CONCLUSION: These results indicate that XB21 plays a role in the plant immune response and in regulation of cell death. The up-regulation of genes controlling 'vesicle-mediated transport' in XB21 overexpression lines is consistent with a functional role for XB21 as an auxilin.

A microbially derived tyrosine-sulfated peptide mimics a plant peptide hormone.

The biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces a sulfated peptide named RaxX, which shares similarity to peptides in the PSY (plant peptide containing sulfated tyrosine) family. We hypothesize that RaxX mimics the growth-stimulating activity of PSY peptides. Root length was measured in Arabidopsis and rice treated with synthetic RaxX peptides. We also used comparative genomic analyses and reactive oxygen species burst assays to evaluate the activity of RaxX and PSY peptides. Here we found that a synthetic sulfated RaxX derivative comprising 13 residues (RaxX13-sY), highly conserved between RaxX and PSY, induces root growth in Arabidopsis and rice in a manner similar to that triggered by PSY. We identified residues that are required for activation of immunity mediated by the rice XA21 receptor but that are not essential for root growth induced by PSY. Finally, we showed that a Xanthomonas strain lacking raxX is impaired in virulence. These findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide.

Silencing of the Rice Gene LRR1 Compromises Rice Xa21 Transcript Accumulation and XA21-Mediated Immunity.

BACKGROUND: The rice immune receptor XA21 confers resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. We previously demonstrated that an auxilin-like protein, XA21 BINDING PROTEIN 21 (XB21), positively regulates resistance to Xoo. RESULTS: To further investigate the function of XB21, we performed a yeast two-hybrid screen. We identified 22 unique XB21 interacting proteins, including LEUCINE-RICH REPEAT PROTEIN 1 (LRR1), which we selected for further analysis. Silencing of LRR1 in the XA21 genetic background (XA21-LRR1Ri) compromises resistance to Xoo compared with control XA21 plants. XA21-LRR1Ri plants have reduced Xa21 transcript levels and reduced expression of genes that serve as markers of XA21-mediated activation. Overexpression of LRR1 is insufficient to alter resistance to Xoo in rice lines lacking XA21. CONCLUSIONS: Taken together, our results indicate that LRR1 is required for wild-type Xa21 transcript expression and XA21-mediated immunity.

Phenotyping of VIGS-mediated gene silencing in rice using a vector derived from a DNA virus.

KEY MESSAGE: Target genes in rice can be optimally silenced if inserted in antisense or hairpin orientation in the RTBV-derived VIGS vector and plants grown at 28 °C and 80% humidity after inoculation. Virus induced gene silencing (VIGS) is a method used to transiently silence genes in dicot as well as monocot plants. For the important monocot species rice, the Rice tungro bacilliform virus (RTBV)-derived VIGS system (RTBV-VIGS), which uses agroinoculation to initiate silencing, has not been standardized for optimal use. Here, using RTBV-VIGS, three sets of conditions were tested to achieve optimal silencing of the rice marker gene phytoene desaturase (pds). The effect of orientation of the insert in the RTBV-VIGS plasmid (sense, antisense and hairpin) on the silencing of the target gene was then evaluated using rice magnesium chelatase subunit H (chlH). Finally, the rice Xa21 gene, conferring resistance against bacterial leaf blight disease (BLB) was silenced using RTBV-VIGS system. In each case, real-time PCR-based assessment indicated approximately 40-80% fall in the accumulation levels of the transcripts of pds, chlH and Xa21. In the case of pds, the appearance of white streaks in the emerging leaves, and for chlH, chlorophyll levels and F v/F m ratio were assessed as phenotypes for silencing. For Xa21, the resistance levels to BLB were assessed by measuring the lesion length and the percent diseased areas of leaves, following challenge inoculation with Xanthomonas oryzae. In each case, the RTBV-MVIGS system gave rise to a discernible phenotype indicating the silencing of the respective target gene using condition III (temperature 28 °C, humidity 80% and 1 mM MES and 20 µM acetosyringone in secondary agrobacterium culture), which revealed the robustness of this gene silencing system for rice.