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.

The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies.

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations.

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.

Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9.

Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.

Whole-Genome Sequencing Identifies a Rice Grain Shape Mutant, gs9-1.

BACKGROUND: Breeding for genes controlling key agronomic traits is an important goal of rice genetic improvement. To gain insight into genes controlling grain morphology, we screened M3 plants derived from 1,000 whole-genome sequenced (WGS) M2 Kitaake mutants to identify lines with altered grain size. RESULTS: In this study, we isolated a mutant, named fast-neutron (FN) 60-4, which exhibits a significant reduction in grain size. We crossed FN60-4 with the parental line Kitaake and analyzed the resulting backcross population. Segregation analysis of 113 lines from the BC2F2 population revealed that the mutant phenotype is controlled by a single semi-dominant locus. Mutant FN60-4 is reduced 20% in plant height and 8.8% in 1000-grain weight compared with Kitaake. FN60-4 also exhibits an 8% reduction in cell number and a 9% reduction in cell length along the vertical axis of the glume. We carried out whole-genome sequencing of DNA pools extracted from segregants with long grains or short grains, and revealed that one gene, LOC_Os09g02650, cosegregated with the grain size phenotype in the BC1F2 and BC2F2 populations. This mutant allele was named grain shape 9-1 (gs9-1). gs9-1 carries a 3-bp deletion that affects two amino acids. This locus is a new allele of the BC12/GDD1/MTD1 gene that encodes a kinesin-like protein involved in cell-cycle progression, cellulose microfibril deposition and gibberellic acid (GA) biosynthesis. The GA biosynthesis-related gene KO2 is down-regulated in gs9-1. The dwarf phenotype of gs9-1 can be rescued by adding exogenous GA3. In contrast to the phenotypes for the other alleles, the gs9-1 is less severe, consistent with the nature of the mutation, which does not disrupt the open reading frame as observed for the other alleles. CONCLUSIONS: In this study, we isolated a mutant, which exhibits altered grain shape and identified the mutated gene, gs9-1. Our study reveals that gs9-1 is a semi-dominant gene that carries a two-amino acid mutation. gs9-1 is allelic to the BC12/GDD1/MTD1 gene involved in GA biosynthesis. These results demonstrate the efficiency and convenience of cloning genes from the whole-genome sequenced Kitaake mutant population to advance investigations into genes controlling key agronomic traits in rice.

Comparative genomics of Saccharomyces cerevisiae natural isolates for bioenergy production.

Lignocellulosic plant material is a viable source of biomass to produce alternative energy including ethanol and other biofuels. However, several factors­including toxic by products from biomass pretreatment and poor fermentation of xylose and other pentose sugars­currently limit the efficiency of microbial biofuel production. To begin to understand the genetic basis of desirable traits, we characterized three strains of Saccharomyces cerevisiae with robust growth in a pretreated lignocellulosic hydrolysate or tolerance to stress conditions relevant to industrial biofuel production, through genome and transcriptome sequencing analysis. All stress resistant strains were highly mosaic, suggesting that genetic admixture may contribute to novel allele combinations underlying these phenotypes. Strain-specific gene sets not found in the lab strain were functionally linked to the tolerances of particular strains. Furthermore,genes with signatures of evolutionary selection were enriched for functional categories important for stress resistance and included stress-responsive signaling factors. Comparison of the strains' transcriptomic responses to heat and ethanol treatment­two stresses relevant to industrial bioethanol production­pointed to physiological processes that were related to particular stress resistance profiles. Many of the genotype-by-environment expression responses occurred at targets of transcription factors with signatures of positive selection, suggesting that these strains have undergone positive selection for stress tolerance. Our results generate new insights into potential mechanisms of tolerance to stresses relevant to biofuel production, including ethanol and heat, present a backdrop for further engineering, and provide glimpses into the natural variation of stress tolerance in wild yeast strains.