Huainian Liu

The evolution and pathogenic mechanisms of the rice sheath blight pathogen

Rhizoctonia solani is a major fungal pathogen of rice (Oryza sativa L.) that causes great yield losses in all rice-growing regions of the world. Here we report the draft genome sequence of the rice sheath blight disease pathogen, R. solani AG1 IA, assembled using next-generation Illumina Genome Analyser sequencing technologies. The genome encodes a large and diverse set of secreted proteins, enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, which probably reflect an exclusive necrotrophic lifestyle. We find few repetitive elements, a closer relationship to Agaricomycotina among Basidiomycetes, and expand protein domains and families. Among the 25 candidate pathogen effectors identified according to their functionality and evolution, we validate 3 that trigger crop defence responses; hence we reveal the exclusive expression patterns of the pathogenic determinants during host infection.

Characterization of Vegetative Insecticidal Protein vip Genes of Bacillus thuringiensis from Sichuan Basin in China

Vegetative insecticidal proteins (Vip), the second generation of insecticides, are produced during the vegetative growth stage of Bacillus thuringiensis (Bt). To perform a systematic study of vip genes in Bt strains from different ecological regions of Sichuan Basin, 1,789 soil samples were collected from this basin, which is situated in the western region of China. The basin has a complicated geomorphology and contains mountains, forests, highlands, hursts, and plains. A total of 2,134 Bt strains have been screened from the 1,789 soil samples. According to the results, three vip-type genes were found in this basin, namely the vip1, vip2, and vip3-type genes. Strains containing vip3-type genes were the most abundant in our collection (67.4%), followed by vip2-type genes (14.6%) and vip1-type genes (8.1%). The three types of vip genes were distributed in most of the regions, but E Mei Mountain and the Ba Lang Mountains only contained vip3 genes in environments with high elevation, low temperature, insufficient oxygen, and abundant snow. Moreover, five novel vip3 genes were found, and these Vip proteins were toxic for Chilo suppressalis. All the results mentioned above suggest that Sichuan Basin is a rich resource for vip genes.

The OsmiR396c‐OsGRF4‐OsGIF1 regulatory module determines grain size and yield in rice

Summary Grain weight is the most important component of rice yield and is mainly determined by grain size, which is generally controlled by quantitative trait loci (QTLs). Although numerous QTLs that regulate grain weight have been identified, the genetic network that controls grain size remains unclear. Herein, we report the cloning and functional analysis of a dominant QTL, grain length and width 2 (GLW2), which positively regulates grain weight by simultaneously increasing grain length and width. The GLW2 locus encodes OsGRF4 (growth‐regulating factor 4) and is regulated by the microRNA miR396c in vivo. The mutation in OsGRF4 perturbs the OsmiR396 target regulation of OsGRF4, generating a larger grain size and enhanced grain yield. We also demonstrate that OsGIF1 (GRF‐interacting factors 1) directly interacts with OsGRF4, and increasing its expression improves grain size. Our results suggest that the miR396c‐OsGRF4‐OsGIF1 regulatory module plays an important role in grain size determination and holds implications for rice yield improvement.

Natural variation in PTB1 regulates rice seed setting rate by controlling pollen tube growth

Grain number, panicle seed setting rate, panicle number and grain weight are the most important components of rice grain yield. To date, several genes related to grain weight, grain number and panicle number have been described in rice. However, no genes regulating the panicle seed setting rate have been functionally characterized. Here we show that the domestication-related POLLEN TUBE BLOCKED 1 (PTB1), a RING-type E3 ubiquitin ligase, positively regulates the rice panicle seed setting rate by promoting pollen tube growth. The natural variation in expression of PTB1 which is affected by the promoter haplotype and the environmental temperature, correlates with the rice panicle seed setting rate. Our results support the hypothesis that PTB1 is an important maternal sporophytic factor of pollen tube growth and a key modulator of the rice panicle seed setting rate. This finding has implications for the improvement of rice yield.

Characterization of cry2-type genes of Bacillus thuringiensis strains from soil-isolated of Sichuan basin, China

Sichuan basin, situated in the west of China, is the fourth biggest basin in China. In order to describe a systematic study of the cry2-type genes resources from Bacillus thuringiensis strains of Sichuan basin, a total of 791 Bacillus thuringiensis strains have been screened from 2650 soil samples in different ecological regions. The method of PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the type of cry2 genes. The results showed that 322 Bacillus thuringiensis strains harbored cry2-type genes and four different RFLP patterns were found. The combination of cry2Aa/cry2Ab genes was the most frequent (90.4%), followed by cry2Aa (6.8%) and cry2Ab alone (2.5%), and only one novel type of cry2 gene was cloned from one isolate (JF19-2). The full-length of this novel gene was obtained by the method of thermal asymmetric interlaced PCR (Tail-PCR), which was designated as cry2Ag1 (GenBank No. ACH91610) by the Bt Pesticide Crystal Protein Nomenclature Committee. In addition, the result of scanning electron microscopic (SEM) observation showed that these strains had erose, spherical, bipyramidal, and square crystal. And the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that these strains harbored about one to three major proteins. These strains exhibited a wide range of insecticidal spectrum toxic to Aedes aegypti (Diptera) and Pieris rapae Linnaeus, 1758 (Lepidoptera). Particularly, JF19-2 contained cry2Ag gene had the highest insecticidal activity. All these researches mentioned above revealed the diversity and particularity of cry2-type gene resources from Bacillus thuringiensis strains in Sichuan basin.

Identification of Genome-Wide Variations among Three Elite Restorer Lines for Hybrid-Rice

Rice restorer lines play an important role in three-line hybrid rice production. Previous research based on molecular tagging has suggested that the restorer lines used widely today have narrow genetic backgrounds. However, patterns of genetic variation at a genome-wide scale in these restorer lines remain largely unknown. The present study performed re-sequencing and genome-wide variation analysis of three important representative restorer lines, namely, IR24, MH63, and SH527, using the Solexa sequencing technology. With the genomic sequence of the Indica cultivar 9311 as the reference, the following genetic features were identified: 267,383 single-nucleotide polymorphisms (SNPs), 52,847 insertion/deletion polymorphisms (InDels), and 3,286 structural variations (SVs) in the genome of IR24; 288,764 SNPs, 59,658 InDels, and 3,226 SVs in MH63; and 259,862 SNPs, 55,500 InDels, and 3,127 SVs in SH527. Variations between samples were also determined by comparative analysis of authentic collections of SNPs, InDels, and SVs, and were functionally annotated. Furthermore, variations in several important genes were also surveyed by alignment analysis in these lines. Our results suggest that genetic variations among these lines, although far lower than those reported in the landrace population, are greater than expected, indicating a complicated genetic basis for the phenotypic diversity of the restorer lines. Identification of genome-wide variation and pattern analysis among the restorer lines will facilitate future genetic studies and the molecular improvement of hybrid rice.

Characterization and expression of cry4Cb1 and cry30Ga1 from Bacillus thuringiensis strain HS18-1.

We characterized a novel Bacillus thuringiensis isolate native to China (HS18-1) that shows a spherical crystal harboring two major proteins of about 70 and 130kDa, and contains three novel cry genes (cry4Cb1, cry30Ga1, cry54-type). Furthermore, the cry4Cb1 and cry30Ga1 genes were expressed in Escherichia coli BL21 (DE3): pLysS. Insecticidal activity tests showed that the cry4Cb1 protein exhibited larvicidal activity against Aedes aegypti (Diptera) and the cry30Ga1 protein was toxic to both A. aegypti and P. xylostella (Lepidoptera).

Re-sequencing and genetic variation identification of a rice line with ideal plant architecture

BackgroundThe ideal plant architecture (IPA) includes several important characteristics such as low tiller numbers, few or no unproductive tillers, more grains per panicle, and thick and sturdy stems. We have developed an indica restorer line 7302R that displays the IPA phenotype in terms of tiller number, grain number, and stem strength. However, its mechanism had to be clarified.FindingsWe performed re-sequencing and genome-wide variation analysis of 7302R using the Solexa sequencing technology. With the genomic sequence of the indica cultivar 9311 as reference, 307 627 SNPs, 57 372 InDels, and 3 096 SVs were identified in the 7302R genome. The 7302R-specific variations were investigated via the synteny analysis of all the SNPs of 7302R with those of the previous sequenced none-IPA-type lines IR24, MH63, and SH527. Moreover, we found 178 168 7302R-specific SNPs across the whole genome and 30 239 SNPs in the predicted mRNA regions, among which 8 517 were Non-syn CDS. In addition, 263 large-effect SNPs that were expected to affect the integrity of encoded proteins were identified from the 7302R-specific SNPs. SNPs of several important previously cloned rice genes were also identified by aligning the 7302R sequence with other sequence lines.ConclusionsOur results provided several candidates account for the IPA phenotype of 7302R. These results therefore lay the groundwork for long-term efforts to uncover important genes and alleles for rice plant architecture construction, also offer useful data resources for future genetic and genomic studies in rice.

OsGIF1 Positively Regulates the Sizes of Stems, Leaves, and Grains in Rice

Growth-regulating factor (GRF) interacting factors (GIFs) are involved in several developmental processes in Arabidopsis. We previously showed that upregulation of OsGIF1 expression improves rice grain size. However, whether OsGIF1 is involved in other developmental processes remains unclear. Here, we report pleiotropic effects of OsGIF1 on rice organ size regulation. Overexpression and functional knock-out via a CRISPR/Cas9 strategy revealed that OsGIF1 not only positively regulates the sizes of rice leaf, stem, and grain but also influences rice reproduction. Expression profiles based on both qRT-PCR and GUS (β-glucuronidase) histochemical staining suggested that OsGIF1 is differentially expressed across various rice tissues, consistent with its roles in regulating the development of multiple rice organs. Additionally, we found that OsGIF1-GFP localized preferentially in the nucleus, which supports its proposed role as a transcriptional cofactor. Further histological analysis suggested that OsGIF1 affected rice organ size possibly by regulating cell size. Our results suggest that OsGIF1 plays important roles in vegetative and reproductive developmental processes, with important implications for rice breeding.

Comprehensive analysis of microRNA-Seq and target mRNAs of rice sheath blight pathogen provides new insights into pathogenic regulatory mechanisms.

MicroRNAs (miRNAs) are ∼22 nucleotide non-coding RNAs that regulate gene expression by targeting mRNAs for degradation or inhibiting protein translation. To investigate whether miRNAs regulate the pathogenesis in necrotrophic fungus Rhizoctonia solani AG1 IA, which causes significant yield loss in main economically important crops, and to determine the regulatory mechanism occurring during pathogenesis, we constructed hyphal small RNA libraries from six different infection periods of the rice leaf. Through sequencing and analysis, 177 miRNA-like small RNAs (milRNAs) were identified, including 15 candidate pathogenic novel milRNAs predicted by functional annotations of their target mRNAs and expression patterns of milRNAs and mRNAs during infection. Reverse transcription-quantitative polymerase chain reaction results for randomly selected milRNAs demonstrated that our novel comprehensive predictions had a high level of accuracy. In our predicted pathogenic protein-protein interaction network of R. solani, we added the related regulatory milRNAs of these core coding genes into the network, and could understand the relationships among these regulatory factors more clearly at the systems level. Furthermore, the putative pathogenic Rhi-milR-16, which negatively regulates target gene expression, was experimentally validated to have regulatory functions by a dual-luciferase reporter assay. Additionally, 23 candidate rice miRNAs that may involve in plant immunity against R. solani were discovered. This first study on novel pathogenic milRNAs of R. solani AG1 IA and the recognition of target genes involved in pathogenicity, as well as rice miRNAs, participated in defence against R. solani could provide new insights into revealing the pathogenic mechanisms of the severe rice sheath blight disease.