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1.  The Role of RaxST, a Prokaryotic Sulfotransferase, and RaxABC, a Putative Type I Secretion System, in Activation of the Rice XA21-Mediated Immune Response 
Scientifica  2014;2014:532816.
Tyrosine sulfation is an important posttranslational modification that determines the outcome of serious diseases in plants and animals. We have recently demonstrated that the plant pathogen Xanthomonas oryzae pv. oryzae (Xoo) carries a functional sulfotransferase (RaxST). raxST is required for activation of rice Xa21-mediated immunity indicating the critical, but unknown, function of raxST in mediating the Xoo/rice interaction. The raxST gene resides in the same operon (raxSTAB) as components of a predicted type I secretion and processing system (RaxA and RaxB). These observations suggest a model where RaxST sulfates a molecule that contains a leader peptide, which is cleaved by the peptidase domain of the RaxB protein and secreted outside the bacterial cell by the RaxABC T1SS.
PMCID: PMC4216712  PMID: 25386383
2.  Lab to Farm: Applying Research on Plant Genetics and Genomics to Crop Improvement 
PLoS Biology  2014;12(6):e1001878.
In this Essay, Pam Ronald describes how basic research advances have been translated into crop improvement, explores some lessons learned, and discusses the potential for plant genetic improvement technologies to contribute to improved food security and agricultural sustainability.
Over the last 300 years, plant science research has provided important knowledge and technologies for advancing the sustainability of agriculture. In this Essay, I describe how basic research advances have been translated into crop improvement, explore some lessons learned, and discuss the potential for current and future contribution of plant genetic improvement technologies to continue to enhance food security and agricultural sustainability.
PMCID: PMC4051633  PMID: 24915201
3.  Tyrosine sulfation in a Gram-negative bacterium 
Nature communications  2012;3:1153.
Tyrosine sulfation, a well-characterized post-translation modification in eukaryotes, has not previously been reported in prokaryotes. Here we demonstrate that the RaxST protein from the Gram-negative bacterium, Xanthomonas oryzae pv. oryzae, is a tyrosine sulfotransferase. We used a newly developed sulfotransferase assay and ultraviolet photodissociation mass spectrometry (UVPD) to demonstrate that RaxST catalyzes sulfation of tyrosine 22 of the Xoo Ax21 (activator of XA21-mediated immunity). These results demonstrate a previously undescribed post-translational modification in a prokaryotic species with implications extending to host immune response and bacterial cell-cell communication system.
PMCID: PMC4305400  PMID: 23093190
4.  The Phylogenetically-Related Pattern Recognition Receptors EFR and XA21 Recruit Similar Immune Signaling Components in Monocots and Dicots 
PLoS Pathogens  2015;11(1):e1004602.
During plant immunity, surface-localized pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs). The transfer of PRRs between plant species is a promising strategy for engineering broad-spectrum disease resistance. Thus, there is a great interest in understanding the mechanisms of PRR-mediated resistance across different plant species. Two well-characterized plant PRRs are the leucine-rich repeat receptor kinases (LRR-RKs) EFR and XA21 from Arabidopsis thaliana (Arabidopsis) and rice, respectively. Interestingly, despite being evolutionary distant, EFR and XA21 are phylogenetically closely related and are both members of the sub-family XII of LRR-RKs that contains numerous potential PRRs. Here, we compared the ability of these related PRRs to engage immune signaling across the monocots-dicots taxonomic divide. Using chimera between Arabidopsis EFR and rice XA21, we show that the kinase domain of the rice XA21 is functional in triggering elf18-induced signaling and quantitative immunity to the bacteria Pseudomonas syringae pv. tomato (Pto) DC3000 and Agrobacterium tumefaciens in Arabidopsis. Furthermore, the EFR:XA21 chimera associates dynamically in a ligand-dependent manner with known components of the EFR complex. Conversely, EFR associates with Arabidopsis orthologues of rice XA21-interacting proteins, which appear to be involved in EFR-mediated signaling and immunity in Arabidopsis. Our work indicates the overall functional conservation of immune components acting downstream of distinct LRR-RK-type PRRs between monocots and dicots.
Author Summary
Pests and diseases cause significant agricultural losses. Plants recognize pathogen-derived molecules via plasma membrane-localized immune receptors (called pattern recognition receptors or PRRs), resulting in pathogen resistance. In recent years, the transfer of PRRs across plant species has emerged as a promising biotechnological approach to improve crop disease resistance. Successful transfers of PRRs suggest that immune signaling components are conserved across plant species. In this study, we demonstrate that the PRR XA21 from the monocot plant rice is functional in the dicot plant Arabidopsis thaliana (Arabidopsis) and that it confers quantitatively enhanced resistance to bacteria. Furthermore, we show that the rice XA21 and the Arabidopsis EFR, which are evolutionary-distant but phylogenetically closely related, recruit similar signaling components for their function, revealing an overall conservation of immune pathways across monocots and dicots. These findings demonstrate evolutionary conservation of downstream signaling from PRRs and indicate that transfer of PRRs is possible between different plant families, but also between monocots and dicots.
PMCID: PMC4301810  PMID: 25607985
5.  Abundance of mixed linkage glucan in mature tissues and secondary cell walls of grasses 
Plant Signaling & Behavior  2013;8(2):e23143.
(1,3; 1,4)-β-D-glucan, also known as mixed linkage glucan (MLG), is a polysaccharide that in flowering plants is unique to the cell walls of grasses and other related members of Poales. MLG is highly abundant in endosperm cell walls, where it is considered a storage carbohydrate. In vegetative tissues, MLG transiently accumulates in the primary cell walls of young, elongating organs. In evolutionary distant species such as Equisetum, MLG accumulates predominantly in old tissues in the stems. Similarly, we have recently shown that rice accumulates a large amount of MLG in mature stems, which prompted us to re-evaluate the hypothesis that MLG is solely related to growth in grass vegetative tissues. Here, we summarize data that confirms the presence of MLG in secondary cell walls and mature tissues in rice and other grasses. Along with these results, we discuss additional evidence indicating a broader role for MLG than previously considered.
PMCID: PMC3657012  PMID: 23299432
(1,3; 1,4)-β-D-glucan; grass cell walls; hemicelluloses; mixed linkage glucan antibody; secondary cell wall
6.  An XA21-Associated Kinase (OsSERK2) Regulates Immunity Mediated by the XA21 and XA3 Immune Receptors 
Molecular Plant  2014;7(5):874-892.
We show that OsSERK2 is a regulator of innate immune signaling mediated by multiple non-RD receptor kinases (RKs) including XA21, XA3, and OsFLS2. OsSerk2-silenced rice lines are impaired in XA21-mediated immunity to Xoo PXO99, XA3-mediated immunity to Xoo PXO86, and OsFLS2-mediated defense responses. Thus, OsSERK2 is broadly involved in PRR-mediated immunity in rice.
The rice XA21 immune receptor kinase and the structurally related XA3 receptor confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast two-hybrid system in a kinase activity-dependent manner. OsSERK2 undergoes bidirectional transphosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. These results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function. Taken together, our findings suggest that the mechanism of OsSERK2-meditated regulation of rice XA21, XA3, and FLS2 differs from that of AtSERK3/BAK1-mediated regulation of Arabidopsis FLS2 and EFR.
PMCID: PMC4064043  PMID: 24482436
immune receptor kinases; somatic embryogenesis receptor kinase (SERK); immunity; Xanthomonas oryzae pv. oryzae; rice.
7.  Three Novel Rice Genes Closely Related to the Arabidopsis IRX9, IRX9L, and IRX14 Genes and Their Roles in Xylan Biosynthesis 
Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx) mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9), Os01g48440 (OsIRX9L), and Os06g47340 (OsIRX14), from glycosyltransferase family 43 as putative orthologs to the putative β-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the over-expression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx) mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase (XylT) activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in XylT activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength.
PMCID: PMC3622038  PMID: 23596448
xylan; irregular xylan mutants; cell walls; type II cell walls; xylosyltransferase
8.  Construction of a rice glycoside hydrolase phylogenomic database and identification of targets for biofuel research 
Glycoside hydrolases (GH) catalyze the hydrolysis of glycosidic bonds in cell wall polymers and can have major effects on cell wall architecture. Taking advantage of the massive datasets available in public databases, we have constructed a rice phylogenomic database of GHs ( This database integrates multiple data types including the structural features, orthologous relationships, mutant availability, and gene expression patterns for each GH family in a phylogenomic context. The rice genome encodes 437 GH genes classified into 34 families. Based on pairwise comparison with eight dicot and four monocot genomes, we identified 138 GH genes that are highly diverged between monocots and dicots, 57 of which have diverged further in rice as compared with four monocot genomes scanned in this study. Chromosomal localization and expression analysis suggest a role for both whole-genome and localized gene duplications in expansion and diversification of GH families in rice. We examined the meta-profiles of expression patterns of GH genes in twenty different anatomical tissues of rice. Transcripts of 51 genes exhibit tissue or developmental stage-preferential expression, whereas, seventeen other genes preferentially accumulate in actively growing tissues. When queried in RiceNet, a probabilistic functional gene network that facilitates functional gene predictions, nine out of seventeen genes form a regulatory network with the well-characterized genes involved in biosynthesis of cell wall polymers including cellulose synthase and cellulose synthase-like genes of rice. Two-thirds of the GH genes in rice are up regulated in response to biotic and abiotic stress treatments indicating a role in stress adaptation. Our analyses identify potential GH targets for cell wall modification.
PMCID: PMC3752443  PMID: 23986771
biofuel; cell wall; database; duplication; glycoside hydrolase; RiceNet; stress
9.  Protein phosphorylation in plant immunity: insights into the regulation of pattern recognition receptor-mediated signaling 
Plants are continuously challenged by pathogens including viruses, bacteria, and fungi. The plant immune system recognizes invading pathogens and responds by activating an immune response. These responses occur rapidly and often involve post-translational modifications (PTMs) within the proteome. Protein phosphorylation is a common and intensively studied form of these PTMs and regulates many plant processes including plant growth, development, and immunity. Most well-characterized pattern recognition receptors (PRRs), including Xanthomonas resistance 21, flagellin sensitive 2, and elongation factor-Tu receptor, possess intrinsic protein kinase activity and regulate downstream signaling through phosphorylation events. Here, we focus on the phosphorylation events of plant PRRs that play important roles in the immune response. We also discuss the role of phosphorylation in regulating mitogen-associated protein kinase cascades and transcription factors in plant immune signaling.
PMCID: PMC3411088  PMID: 22876255
EFR; FLS2; pattern recognition receptor; plant immunity; post-translational modifications; protein phosphorylation; XA21
10.  Interaction specificity and coexpression of rice NPR1 homologs 1 and 3 (NH1 and NH3), TGA transcription factors and Negative Regulator of Resistance (NRR) proteins 
BMC Genomics  2014;15(1):461.
The nonexpressor of pathogenesis-related genes 1, NPR1 (also known as NIM1 and SAI1), is a key regulator of SA-mediated systemic acquired resistance (SAR) in Arabidopsis. In rice, the NPR1 homolog 1 (NH1) interacts with TGA transcriptional regulators and the Negative Regulator of Resistance (NRR) protein to modulate the SAR response. Though five NPR1 homologs (NHs) have been identified in rice, only NH1 and NH3 enhance immunity when overexpressed. To understand why NH1 and NH3, but not NH2, NH4, or NH5, contribute to the rice immune response, we screened TGA transcription factors and NRR-like proteins for interactions specific to NH1 and NH3. We also examined their co-expression patterns using publicly available microarray data.
We tested five NHs, four NRR homologs (RHs), and 13 rice TGA proteins for pair-wise protein interactions using yeast two-hybrid (Y2H) and split YFP assays. A survey of 331 inter-family interactions revealed a broad, complex protein interaction network. To investigate preferred interaction partners when all three families of proteins were present, we performed a bridged split YFP assay employing YFPN-fused TGA, YFPC-fused RH, and NH proteins without YFP fusions. We found 64 tertiary interactions mediated by NH family members among the 120 sets we examined. In the yeast two-hybrid assay, each NH protein was capable of interacting with most TGA and RH proteins. In the split YFP assay, NH1 was the most prevalent interactor of TGA and RH proteins, NH3 ranked the second, and NH4 ranked the third. Based on their interaction with TGA proteins, NH proteins can be divided into two subfamilies: NH1, NH2, and NH3 in one family and NH4 and NH5 in the other.
In addition to evidence of overlap in interaction partners, co-expression analyses of microarray data suggest a correlation between NH1 and NH3 expression patterns, supporting their common role in rice immunity. However, NH3 is very tightly co-expressed with RH1 and RH2, while NH1 is strongly, inversely co-expressed with RH proteins, representing a difference between NH1 and NH3 expression patterns.
Our genome-wide surveys reveal that each rice NH protein can partner with many rice TGA and RH proteins and that each NH protein prefers specific interaction partners. NH1 and NH3 are capable of interacting strongly with most rice TGA and RH proteins, whereas NH2, NH4, and NH5 have weaker, limited interaction with TGA and RH proteins in rice cells. We have identified rTGA2.1, rTGA2.2, rTGA2.3, rLG2, TGAL2 and TGAL4 proteins as the preferred partners of NH1 and NH3, but not NH2, NH4, or NH5. These TGA proteins may play an important role in NH1- and NH3-mediated immune responses. In contrast, NH4 and NH5 preferentially interact with TGAL5, TGAL7, TGAL8 and TGAL9, which are predicted to be involved in plant development.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-461) contains supplementary material, which is available to authorized users.
PMCID: PMC4094623  PMID: 24919709
SAR; Salicylic acid; NPR1; NH1; TGA; Protein interaction; Yeast two-hybrid; Split YFP; Co-expression; Innate immunity; Bridged split YFP; RH
11.  Differential Requirement of Oryza sativa RAR1 in Immune Receptor-Mediated Resistance of Rice to Magnaporthe oryzae 
Molecules and Cells  2013;35(4):327-334.
The required for Mla12 resistance (RAR1) protein is essential for the plant immune response. In rice, a model monocot species, the function of Oryza sativa RAR1 (OsRAR1) has been little explored. In our current study, we characterized the response of a rice osrar1 T-DNA insertion mutant to infection by Magnaporthe oryzae, the causal agent of rice blast disease. osrar1 mutants displayed reduced resistance compared with wild type rice when inoculated with the normally virulent M. oryzae isolate PO6-6, indicating that OsRAR1 is required for an immune response to this pathogen. We also investigated the function of Os-RAR1 in the resistance mechanism mediated by the immune receptor genes Pib and Pi5 that encode nucleotide binding-leucine rich repeat (NB-LRR) proteins. We inoculated progeny from Pib/osrar1 and Pi5/osrar1 heterozygous plants with the avirulent M. oryzae isolates, race 007 and PO6-6, respectively. We found that only Pib-mediated resistance was compromised by the osrar1 mutation and that the introduction of the OsRAR1 cDNA into Pib/osrar1 rescued Pib-mediated resistance. These results indicate that OsRAR1 is required for Pib-mediated resistance but not Pi5-mediated resistance to M. oryzae.
PMCID: PMC3887888  PMID: 23563801
immune receptor; NB-LRR; OsRAR1; resistance; rice
13.  Retraction: Small Protein-Mediated Quorum Sensing in a Gram-Negative Bacterium 
PLoS ONE  2013;8(9):10.1371/annotation/880a72e1-9cf3-45a9-bf1c-c74ccb73fd35.
PMCID: PMC3789838  PMID: 24098628
14.  Identification of a host 14-3-3 Protein that Interacts with Xanthomonas effector AvrRxv 
AvrRxv is a member of a family of pathogen effectors present in pathogens of both plant and mammalian species. Xanthomonas campestris pv. vesicatoria strains carrying AvrRxv induce a hypersensitive response (HR) in the tomato cultivar Hawaii 7998. Using a yeast two-hybrid screen, we identified a 14-3-3 protein from tomato that interacts with AvrRxv called AvrRxv Interactor 1 (ARI1). The interaction was confirmed in vitro with affinity chromatography. Using mutagenesis, we identified a 14-3-3-binding domain in AvrRxv and demonstrated that a mutant in that domain showed concomitant loss of interaction with ARI1 and HR-inducing activity in tomato. These results demonstrate that the AvrRxv bacterial effector recruits 14-3-3 proteins for its function within host cells. AvrRxv homologues YopP and YopJ from Yersinia do not have AvrRxv-specific HR-inducing activity when delivered into tomato host cells by Agrobacterium. Although YopP itself cannot induce HR, its C-terminal domain containing the catalytic residues can replace that of AvrRxv in an AvrRxv-YopP chimera for HR-inducing activity. Phylogenetic analysis indicates that the sequences encoding the C-termini of family members are evolving independently from those encoding the N-termini. Our results support a model in which there are three functional domains in proteins of the family, translocation, interaction, and catalytic.
PMCID: PMC3142867  PMID: 21796232
avirulence protein; yeast two hybrid interaction screen; effector; hypersensitive response (HR); Xanthomonas campestris pv. vesicatoria; Yersinia enterocolitica; Solanum esculentum
15.  Comparative analysis of protein-protein interactions in the defense response of rice and wheat 
BMC Genomics  2013;14:166.
Despite the importance of wheat as a major staple crop and the negative impact of diseases on its production worldwide, the genetic mechanisms and gene interactions involved in the resistance response in wheat are still poorly understood. The complete sequence of the rice genome has provided an extremely useful parallel road map for genetic and genomics studies in wheat. The recent construction of a defense response interactome in rice has the potential to further enhance the translation of advances in rice to wheat and other grasses. The objective of this study was to determine the degree of conservation in the protein-protein interactions in the rice and wheat defense response interactomes. As entry points we selected proteins that serve as key regulators of the rice defense response: the RAR1/SGT1/HSP90 protein complex, NPR1, XA21, and XB12 (XA21 interacting protein 12).
Using available wheat sequence databases and phylogenetic analyses we identified and cloned the wheat orthologs of these four rice proteins, including recently duplicated paralogs, and their known direct interactors and tested 86 binary protein interactions using yeast-two-hybrid (Y2H) assays. All interactions between wheat proteins were further tested using in planta bimolecular fluorescence complementation (BiFC). Eighty three percent of the known rice interactions were confirmed when wheat proteins were tested with rice interactors and 76% were confirmed using wheat protein pairs. All interactions in the RAR1/SGT1/ HSP90, NPR1 and XB12 nodes were confirmed for the identified orthologous wheat proteins, whereas only forty four percent of the interactions were confirmed in the interactome node centered on XA21. We hypothesize that this reduction may be associated with a different sub-functionalization history of the multiple duplications that occurred in this gene family after the divergence of the wheat and rice lineages.
The observed high conservation of interactions between proteins that serve as key regulators of the rice defense response suggests that the existing rice interactome can be used to predict interactions in wheat. Such predictions are less reliable for nodes that have undergone a different history of duplications and sub-functionalization in the two lineages.
PMCID: PMC3602203  PMID: 23496930
16.  Reduced expression of glycolate oxidase leads to enhanced disease resistance in rice 
PeerJ  2013;1:e28.
Glycolate oxidase (GLO) is a key enzyme in photorespiration, catalyzing the oxidation of glycolate to glyoxylate. Arabidopsis GLO is required for nonhost defense responses to Pseudomonas syringae and for tobacco Pto/AvrPto-mediated defense responses. We previously described identification of rice GLO1 that interacts with a glutaredoxin protein, which in turn interacts with TGA transcription factors. TGA transcription factors are well known to participate in NPR1/NH1-mediated defense signaling, which is crucial to systemic acquired resistance in plants. Here we demonstrate that reduction of rice GLO1 expression leads to enhanced resistance to Xanthomonas oryzae pv oryzae (Xoo). Constitutive silencing of GLO1 leads to programmed cell death, resulting in a lesion-mimic phenotype and lethality or reduced plant growth and development, consistent with previous reports. Inducible silencing of GLO1, employing a dexamethasone-GVG (Gal4 DNA binding domain-VP16 activation domain-glucocorticoid receptor fusion) inducible system, alleviates these detrimental effects. Silencing of GLO1 results in enhanced resistance to Xoo, increased expression of defense regulators NH1, NH3, and WRKY45, and activation of PR1 expression.
PMCID: PMC3628735  PMID: 23638363
Photorespiration; Glycolate oxidase; Disease resistance; NH1; NPR1; Glutaredoxin; Hydrogen peroxide; WRKY45
17.  Innate immunity in rice 
Trends in plant science  2011;16(8):451-459.
Advances in studies of rice innate immunity have led to the identification and characterization of host sensors encoding receptor kinases that perceive conserved microbial signatures. The non-RD domain, a newly recognized hallmark of these receptor kinases is highly expanded in rice (Oryza sativa) compared with Arabidopsis (Arabidopsis thaliana). Researchers have also identified a diverse array of microbial effectors from bacterial and fungal pathogens that triggers immune responses upon perception. These include both, effectors that indirectly target host Nucleotide binding site/Leucine rice repeat (NBS-LRR) proteins and transcription activator-like (TAL) effectors that directly bind promoters of host genes. Here we review the recognition and signaling events that govern rice innate immunity.
PMCID: PMC3152591  PMID: 21602092
18.  A Genome-Wide Survey of Switchgrass Genome Structure and Organization 
PLoS ONE  2012;7(4):e33892.
The perennial grass, switchgrass (Panicum virgatum L.), is a promising bioenergy crop and the target of whole genome sequencing. We constructed two bacterial artificial chromosome (BAC) libraries from the AP13 clone of switchgrass to gain insight into the genome structure and organization, initiate functional and comparative genomic studies, and assist with genome assembly. Together representing 16 haploid genome equivalents of switchgrass, each library comprises 101,376 clones with average insert sizes of 144 (HindIII-generated) and 110 kb (BstYI-generated). A total of 330,297 high quality BAC-end sequences (BES) were generated, accounting for 263.2 Mbp (16.4%) of the switchgrass genome. Analysis of the BES identified 279,099 known repetitive elements, >50,000 SSRs, and 2,528 novel repeat elements, named switchgrass repetitive elements (SREs). Comparative mapping of 47 full-length BAC sequences and 330K BES revealed high levels of synteny with the grass genomes sorghum, rice, maize, and Brachypodium. Our data indicate that the sorghum genome has retained larger microsyntenous regions with switchgrass besides high gene order conservation with rice. The resources generated in this effort will be useful for a broad range of applications.
PMCID: PMC3325252  PMID: 22511929
19.  Two New Complete Genome Sequences Offer Insight into Host and Tissue Specificity of Plant Pathogenic Xanthomonas spp.▿† 
Journal of Bacteriology  2011;193(19):5450-5464.
Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.
PMCID: PMC3187462  PMID: 21784931
20.  A rice transient assay system identifies a novel domain in NRR required for interaction with NH1/OsNPR1 and inhibition of NH1-mediated transcriptional activation 
Plant Methods  2012;8:6.
Arabidopsis NPR1 is a master regulator of systemic acquired resistance. NPR1 binds to TGA transcription factors and functions as a transcriptional co-activator. In rice, NH1/OsNPR1 functions to enhance innate immunity. NRR disrupts NH1 function, when over-expressed.
We have established a rice transient protoplast assay to demonstrate that NH1 is a transcriptional co-activator and that NRR represses NH1-mediated activation. We identified three NRR homologues (RH1, RH2, and RH3). RH1 and RH3, but not RH2, also effectively repress NH1-mediated transcriptional activation. NRR, RH1, RH2, and RH3 share sequence similarity in a region beyond the previously identified NPR1-interacting domain. This region is required for strong interaction with NH1. A double point mutation, W66A/F70A, in this novel NH1-interacting domain severely reduces interaction with NH1. Mutation W66A/F70A also greatly reduces the ability of NRR to repress NH1-mediated activation. RH2 carries a deviation (amino acids AV) in this region as compared to consensus sequences (amino acids ED) among NRR, RH1, and RH3. A substitution (AV to ED) in RH2 results in strong binding of mutant RH2ED to NH1 and effective repression of NH1-mediated activation.
The protoplast-based transient system can be used to dissect protein domains associated with their functions. Our results demonstrate that the ability of NRR and its homologues to repress NH1-mediated transcriptional activation is tightly correlated with their ability to bind to NH1. Furthermore, a sequence is identified as a novel NH1-interacting domain. Importantly, this novel sequence is widely present in plant species, from cereals to castor bean plants, to poplar trees, to Arabidopsis, indicating its significance in plants.
PMCID: PMC3297495  PMID: 22353606
21.  Small Protein-Mediated Quorum Sensing in a Gram-Negative Bacterium 
PLoS ONE  2011;6(12):e29192.
The rice XA21 pattern recognition receptor binds a type I secreted sulfated peptide, called axYS22, derived from the Ax21 (activator of XA21-mediated immunity) protein. The conservation of Ax21 in all sequenced Xanthomonas spp. and closely related genera suggests that Ax21 serves a key biological function. Here we show that the predicted N-terminal sequence of Ax21 is cleaved prior to secretion outside the cell and that mature Ax21 serves as a quorum sensing (QS) factor in Xanthomonas oryzae pv. oryzae. Ax21-mediated QS controls motility, biofilm formation and virulence. We provide genetic evidence that the Xoo RaxH histidine kinase serves as the bacterial receptor for Ax21. This work establishes a critical role for small protein-mediated QS in a Gram-negative bacterium.
PMCID: PMC3236232  PMID: 22174954
22.  Protein abundances are more conserved than mRNA abundances across diverse taxa 
Proteomics  2010;10(23):4209-4212.
Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post-transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large-scale protein and mRNA expression datasets from seven different species: two bacteria, yeast, nematode, fly, human, and plant. We find that steady-state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge.
PMCID: PMC3113407  PMID: 21089048
23.  Ectopic expression of rice Xa21 overcomes developmentally controlled resistance to Xanthomonas oryzae pv. oryzae 
Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) activates the innate immune response. The rice PRR, XA21, confers robust resistance at adult stages to most strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Seedlings are still easily infected by Xoo, causing severe yield losses. Here we report that Xa21 is induced by Xoo infection and that ectopic expression of Xa21 confers resistance at three leaf stage (three-week-old), overcoming the developmental limitation of XA21-mediated resistance. Ectopic expression of Xa21 also up-regulates a larger set of defense-related genes as compared to Xa21 driven by the native promoter. These results indicate that altered regulation of Xa21 expression is useful for developing enhanced resistance to Xoo at multiple developmental stages.
PMCID: PMC2976559  PMID: 21076626
Oryza sativa; pathogen-associated molecular pattern; pattern recognition receptor; XA21; Xanthomonas oryzae pv. oryzae
24.  Combining multivariate analysis and monosaccharide composition modeling to identify plant cell wall variations by Fourier Transform Near Infrared spectroscopy 
Plant Methods  2011;7:26.
We outline a high throughput procedure that improves outlier detection in cell wall screens using FT-NIR spectroscopy of plant leaves. The improvement relies on generating a calibration set from a subset of a mutant population by taking advantage of the Mahalanobis distance outlier scheme to construct a monosaccharide range predictive model using PLS regression. This model was then used to identify specific monosaccharide outliers from the mutant population.
PMCID: PMC3168417  PMID: 21851585
near infrared spectroscopy; cell wall; hemicellulose; multivariate analysis; mutant screen; pls modeling
25.  Elucidation of XA21-mediated Innate Immunity 
Cellular microbiology  2010;12(8):1017-1025.
In the early 1970s, the Xa21 gene from the wild rice species Oryza longistaminata, drew attention of rice breeders because of its broad-spectrum resistance to diverse strains of a serious bacterial disease of rice in Asia and Africa, called bacterial blight disease, caused by the Gram-negative bacterium, Xanthomonas oryzae pv. oryzae (Xoo). In 1995, we isolated the gene controlling this resistance and in 2009 demonstrated that XA21 recognizes a highly conserved peptide, called Ax21 (activator of XA21-mediated immunity). Tyrosine sulfation of Ax21 is required for recognition by rice XA21. A decade of genetic, molecular, and biochemical studies have uncovered key components of the XA21-mediated signaling cascade. Ax21 recognition by XA21 at the cell surface induces phosphorylation–mediated events, which are predicted to alter subcellular localization and/or DNA-binding activity of a WRKY family of transcription factors. Because XA21 is representative of the large number of predicted pattern recognition receptors (PRRs) in rice (328), Arabidopsis (35) and other plant species, further characterization of XA21-mediated signaling pathways will contribute to elucidation of these important defense responses.
PMCID: PMC2906629  PMID: 20590657

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