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 riceblast 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.

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.

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 broadspectrum disease resistance and the evolutionary mechanisms of blast resistance gene clusters in rice.

SUMMARYThe transient assay system based on mesophyll or cultured cell-derived protoplasts has been exploited in several plant species and has become a powerful tool for rapid gene functional analysis and biochemical manipulations. However, the system has not been widely used in rice owing to the difficulties in large-scale isolation of viable rice protoplasts from leaves or suspension-cultured cells. Here, we describe a significantly improved method to isolate a large number of protoplasts from stem and sheath tissues of both young and mature plants. High-level coexpression of multiple constructs and efficient suppression of exogenous and endogenous genes were observed in the stemand sheath-derived protoplasts. A transient green fluorescent protein and luciferase-based reporter system for defence-related genes expression analysis has been established, which is useful for screening and characterizing genes involved in rice defence signalling pathways. Furthermore, a protoplast-based bimolecular fluorescence complementation (BiFC) system for the detection of protein-protein interactions in living rice cells was developed. The YFP complementation of two split-YFP halves mediated by homodimerization of the GUS and SPIN1, a cell-death related protein, was observed in transfected protoplasts. In combination with genetic, genomic and proteomic approaches, the established versatile protoplast transient assay system will facilitate largescale functional analysis of defence-related genes in rice.

Rice feeds more than half of the world's population. Rice blast, caused by the fungal pathogen Magnaporthe oryzae, and bacterial blight, caused by the bacterial pathogen Xanthomonas oryzae pv. oryzae, are major constraints to rice production worldwide. Genome sequencing and extensive molecular analysis has led to the identification of many new pathogen-associated molecular patterns (PAMPs) and avirulence and virulence effectors in both pathogens, as well as effector targets and receptors in the rice host. Characterization of these effectors, host targets, and resistance genes has provided new insight into innate immunity in plants. Some of the new findings, such as the binding activity of X. oryzae transcriptional activator-like (TAL) effectors to specific rice genomic sequences, are being used for the development of effective disease control methods and genome modification tools. This review summarizes the recent progress toward understanding the recognition and signaling events that govern rice innate immunity.

Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability.DOI: http://dx.doi.org/10.7554/eLife.00354.001

Many plant mutants develop spontaneous lesions that resemble disease symptoms in the absence of pathogen attack. In several pathosystems, lesion mimic mutations have been shown to be involved in programmed cell death, which in some instances leads to enhanced disease resistance to multiple pathogens. We investigated the relationship between spontaneous cell death and disease resistance in rice with nine mutants with a range of lesion mimic phenotypes. All nine mutations are controlled by recessive genes and some of these mutants have stunted growth and other abnormal characteristics. The lesion mimics that appeared on the leaves of these mutants were caused by cell death as measured by trypan blue staining. Activation of six defense-related genes was observed in most of the mutants when the mimic lesions developed. Four mutants exhibited significant enhanced resistance to rice blast. One of the mutants, spl11, confers non-race-specific resistance not only to blast but also to bacterial blight. The level of resistance in the spl11 mutant to the two pathogens correlates with the defense-related gene expression and lesion development on the leaves. The results suggest that some lesion mimic mutations in rice may be involved in disease resistance, and cloning of these genes may provide a clue to developing broad-spectrum resistance to diverse pathogens.

IR64, the most widely grown indica rice 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.