Guo-Liang Wang

A Receptor Kinase-Like Protein Encoded by the Rice Disease Resistance Gene, Xa21

The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by positional cloning. Fifty transgenic rice plants carrying the cloned Xa21 gene display high levels of resistance to the pathogen. The sequence of the predicted protein, which carries both a leucine-rich repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice.

R gene expression induced by a type-III effector triggers disease resistance in rice.

Disease resistance (R) genes in plants encode products that specifically recognise incompatible pathogens and trigger a cascade of events leading to disease resistance in the host plant. R-gene specificity is dictated by both host R genes and cognate avirulence (avr) genes in pathogens. However, the basis of gene-for-gene specificity is not well understood. Here, we report the cloning of the R gene Xa27 from rice and the cognate avr gene avrXa27 from Xanthomonas oryzae pv. oryzae. Resistant and susceptible alleles of Xa27 encode identical proteins. However, expression of only the resistant allele occurs when a rice plant is challenged by bacteria harbouring avrXa27, whose product is a nuclear localized type-III effector. Induction of Xa27 occurs only in the immediate vicinity of infected tissue, whereas ectopic expression of Xa27 resulted in resistance to otherwise compatible strains of the pathogen. Thus Xa27 specificity towards incompatible pathogens involves the differential expression of the R gene in the presence of the AvrXa27 effector.

The Broad-Spectrum Blast Resistance Gene Pi9 Encodes a Nucleotide-Binding Site–Leucine-Rich Repeat Protein and Is a Member of a Multigene Family in Rice

The broad-spectrum rice blast resistance gene Pi9 was cloned using a map-based cloning strategy. Sequencing of a 76-kb bacterial artificial chromosome (BAC) contig spanning the Pi9 locus led to identification of six tandemly arranged resistance-like genes with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) (Nbs1-Pi9–Nbs6-Pi9). Analysis of selected Pi9 deletion mutants and transformation of a 45-kb fragment from the BAC contig into the susceptible rice cultivar TP309 narrowed down Pi9 to the candidate genes Nbs2-Pi9 and Nbs3-Pi9. Disease evaluation of the transgenic lines carrying the individual candidate genes confirmed that Nbs2-Pi9 is the Pi9 gene. Sequence comparison analysis revealed that the six paralogs at the Pi9 locus belong to four classes and gene duplication might be one of the major evolutionary forces contributing to the formation of the NBS–LRR gene cluster. Semiquantitative reverse transcriptase (RT)–PCR analysis showed that Pi9 was constitutively expressed in the Pi9-resistant plants and was not induced by blast infection. The cloned Pi9 gene provides a starting point to elucidate the molecular basis of the broad-spectrum disease resistance and the evolutionary mechanisms of blast resistance gene clusters in rice.

Locating genes associated with root morphology and drought avoidance in rice via linkage to molecular markers

This research was undertaken to identify and map quantitative trait loci (QTLs) associated with five parameters of rice root morphology and to determine if these QTLs are located in the same chromosomal regions as QTLs associated with drought avoidance/tolerance. Root thickness, root:shoot ratio, root dry weight per tiller, deep root dry weight per tiller, and maximum root length were measured in three replicated experiments (runs) of 203 recombinant inbred lines grown in a greenhouse. The lines were from a cross between indica cultivar Co39 andjaponica cultivar Moroberekan. The 203 RI lines were also grown in three replicated field experiments where they were drought-stressed at the seedling, early vegetative, and late-vegetative growth stage and assigned a visual rating based on leaf rolling as to their degree of drought avoidance/tolerance. The QTL analysis of greenhouse and field data was done using single-marker analysis (ANOVA) and interval analysis (Mapmaker QTL). Most QTLs that were identified were associated with root thickness, root/shoot ratio, and root dry weight per tiller, and only a few with deep root weight. None were reliably associated with maximum root depth due to genotype-by-experiment interaction. Root thickness and root dry weight per tiller were the characters found to be the least influenced by environmental differences between greenhouse runs. Correlations of root parameters measured in greenhouse experiments with field drought avoidance/tolerance were significant but not highly predictive. Twelve of the fourteen chromosomal regions containing putative QTLs associated with field drought avoidance/tolerance also contained QTLs associated with root morphology. Thus, selecting for Moroberekan alleles at marker loci associated with the putative root QTLs identified in this study may be an effective strategy for altering the root phenotype of rice towards that commonly associated with drought-resistant cultivars.

Xa21D Encodes a Receptor-like Molecule with a Leucine-Rich Repeat Domain That Determines Race-Specific Recognition and Is Subject to Adaptive Evolution

The rice Xa21 gene confers resistance to Xanthomonas oryzae pv oryzae in a race-specific manner. Analysis of the inheritance patterns and resistance spectra of transgenic plants carrying six Xa21 gene family members indicated that one member, designated Xa21D, displayed a resistance spectrum identical to that observed for Xa21 but conferred only partial resistance. Xa21D encodes a receptor-like protein carrying leucine-rich repeat (LRR) motifs in the presumed extracellular domain. The Xa21D transcript terminates shortly after the stop codon introduced by the retrotransposon Retrofit. Comparison of nucleotide substitutions in the LRR coding regions of Xa21 and Xa21D provided evidence of adaptive selection. Both functional and evolutionary evidence indicates that the Xa21D LRR domain controls race-specific pathogen recognition.

Evolution of the rice Xa21 disease resistance gene family.

The rice disease resistance gene Xa21, encoding a receptor-like kinase, is a member of a multigene family. Sequence analysis of seven family members revealed two distinct classes of genes. One member from each class encodes a receptor kinase-like open reading frame. The other five members encode truncated open reading frames of the predicted receptor kinase. A highly conserved 233-bp sequence (HC) was also identified among the seven family members. Recombination at the HC region between family members apparently resulted in the precise swapping of promoter regions. Large sequence duplications were generated by a presumed unequal crossover event in intergenic regions. Insertions of transposon-like sequences truncated two of the predicted open reading frames. A model for amplification and diversification of the Xa21 gene family is presented.

The Eight Amino-Acid Differences Within Three Leucine-Rich Repeats Between Pi2 and Piz-t Resistance Proteins Determine the Resistance Specificity to Magnaporthe grisea

The rice blast resistance (R) genes Pi2 and Piz-t confer broad-spectrum resistance against different sets of Magnaporthe grisea isolates. We first identified the Pi2 gene using a map-based cloning strategy. The Pi2 gene is a member of a gene cluster comprising nine gene members (named Nbs1-Pi2 to Nbs9-Pi2) and encodes a protein with a nucleotide-binding site and leucine-rich repeat (LRR) domain. Fine genetic mapping, molecular characterization of the Pi2 susceptible mutants, and complementation tests indicated that Nbs4-Pi2 is the Pi2 gene. The Piz-t gene, a Pi2 allele in the rice cultivar Toride 1, was isolated based on the Pi2 sequence information. Complementation tests confirmed that the family member Nbs4-Piz-t is Piz-t. Sequence comparison revealed that only eight amino-acid changes, which are confined within three consecutive LRR, differentiate Piz-t from Pi2. Of the eight variants, only one locates within the xxLxLxx motif. A reciprocal exchange of the single amino acid between Pi2 and Piz-t did not convert the resistance specificity to each other but, rather, abolished the function of both resistance proteins. These results indicate that the single amino acid in the xxLxLxx motif may be critical for maintaining the recognition surface of Pi2 and Piz-t to their respective avirulence proteins.

Spotted leaf11, a Negative Regulator of Plant Cell Death and Defense, Encodes a U-Box/Armadillo Repeat Protein Endowed with E3 Ubiquitin Ligase Activity

The rice (Oryza sativa) spotted leaf11 (spl11) mutant was identified from an ethyl methanesulfonate–mutagenized indica cultivar IR68 population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice fungal and bacterial pathogens. Here, we have isolated Spl11 via a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection. The predicted SPL11 protein contains both a U-box domain and an armadillo (ARM) repeat domain, which were demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein–protein interactions, respectively. Amino acid sequence comparison indicated that the similarity between SPL11 and other plant U-box-ARM proteins is mostly restricted to the U-box and ARM repeat regions. A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. Expression analysis indicated that Spl11 is induced in both incompatible and compatible rice–blast interactions. In vitro ubiquitination assay indicated that the SPL11 protein possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting a role of the ubiquitination system in the control of plant cell death and defense.

Chemical- and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics

IR64, the most widely grown indicarice in South and Southeast Asia, possesses many positive agronomic characteristics (e.g., wide adaptability, high yield potential, tolerance to multiple diseases and pests, and good eating quality,) that make it an ideal genotype for identifying mutational changes in traits of agronomic importance. We have produced a large collection of chemical and irradiation-induced IR64 mutants with different genetic lesions that are amenable to both forward and reverse genetics. About 60,000 IR64 mutants have been generated by mutagenesis using chemicals (diepoxybutane and ethylmethanesulfonate) and irradiation (fast neutron and gamma ray). More than 38,000 independent lines have been advanced to M4 generation enabling evaluation of quantitative traits by replicated trials. Morphological variations at vegetative and reproductive stages, including plant architecture, growth habit, pigmentation and various physiological characters, are commonly observed in the four mutagenized populations. Conditional mutants such as gain or loss of resistance to blast, bacterial blight, and tungro disease have been identified at frequencies ranging from 0.01% to 0.1%. Results from pilot experiments indicate that the mutant collections are suitable for reverse genetics through PCR-detection of deletions and TILLING. Furthermore, deletions can be detected using oligomer chips suggesting a general technique to pinpoint deletions when genome-wide oligomer chips are broadly available. M4 mutant seeds are available for users for screening of altered response to multiple stresses. So far, more than 15,000 mutant lines have been distributed. To facilitate broad usage of the mutants, a mutant database has been constructed in the International Rice Information System (IRIS; http: //www.iris.irri.org) to document the phenotypes and gene function discovered by users.

A Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics

With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.