Shigui Li

ABCG15 Encodes an ABC Transporter Protein, and is Essential for Post-Meiotic Anther and Pollen Exine Development in Rice

In flowering plants, anther and pollen development is critical for male reproductive success. The anther cuticle and pollen exine play an essential role, and in many cereals, such as rice, orbicules/ubisch bodies are also thought to be important for pollen development. The formation of the anther cuticle, exine and orbicules is associated with the biosynthesis and transport of wax, cutin and sporopollenin components. Recently, progress has been made in understanding the biosynthesis of sporopollenin and cutin components in Arabidopsis and rice, but less is known about the mechanisms by which they are transported to the sites of deposition. Here, we report that the rice ATP-binding cassette (ABC) transporter, ABCG15, is essential for post-meiotic anther and pollen development, and is proposed to play a role in the transport of rice anther cuticle and sporopollenin precursors. ABCG15 is highly expressed in the tapetum at the young microspore stage, and the abcg15 mutant exhibits small, white anthers lacking mature pollen, lipidic cuticle, orbicules and pollen exine. Gas chromatography-mass spectrometry (GC-MS) analysis of the abcg15 anther cuticle revealed significant reductions in a number of wax components and aliphatic cutin monomers. The expression level of genes involved in lipid metabolism in the abcg15 mutant was significantly different from their levels in the wild type, possibly due to perturbations in the homeostasis of anther lipid metabolism. Our study provides new insights for understanding the molecular mechanism of the formation of the anther cuticle, orbicules and pollen wall, as well as the machinery for lipid metabolism in rice anthers.

Sequencing and de novo assembly of a near complete indica rice genome

A high-quality reference genome is critical for understanding genome structure, genetic variation and evolution of an organism. Here we report the de novo assembly of an indica rice genome Shuhui498 (R498) through the integration of single-molecule sequencing and mapping data, genetic map and fosmid sequence tags. The 390.3 Mb assembly is estimated to cover more than 99% of the R498 genome and is more continuous than the current reference genomes of japonica rice Nipponbare (MSU7) and Arabidopsis thaliana (TAIR10). We annotate high-quality protein-coding genes in R498 and identify genetic variations between R498 and Nipponbare and presence/absence variations by comparing them to 17 draft genomes in cultivated rice and its closest wild relatives. Our results demonstrate how to de novo assemble a highly contiguous and near-complete plant genome through an integrative strategy. The R498 genome will serve as a reference for the discovery of genes and structural variations in rice.

Integrated analysis of phenome, genome, and transcriptome of hybrid rice uncovered multiple heterosis-related loci for yield increase.

Significance Because of its practical importance and scientific significance, heterosis (hybrid vigor) is an interesting topic for both breeders and biologists. However, although heterosis has been applied successfully to increase crop yields, the molecular mechanisms involved remain obscure. In this study, using an integrative approach, we found that multiple quantitative trait loci (QTLs) cumulatively drive yield heterosis in hybrid rice by regulating two grain-yield component traits in which the RH8 (rice heterosis 8) gene plays a major role. Our research highlights the importance of integrative methods to uncover the molecular mechanism of heterosis and thus pave a way toward revealing the molecular mechanisms in rice heterosis in detail. Hybrid rice is the dominant form of rice planted in China, and its use has extended worldwide since the 1970s. It offers great yield advantages and has contributed greatly to the world’s food security. However, the molecular mechanisms underlying heterosis have remained a mystery. In this study we integrated genetics and omics analyses to determine the candidate genes for yield heterosis in a model two-line rice hybrid system, Liang-you-pei 9 (LYP9) and its parents. Phenomics study revealed that the better parent heterosis (BPH) of yield in hybrid is not ascribed to BPH of all the yield components but is specific to the BPH of spikelet number per panicle (SPP) and paternal parent heterosis (PPH) of effective panicle number (EPN). Genetic analyses then identified multiple quantitative trait loci (QTLs) for these two components. Moreover, a number of differentially expressed genes and alleles in the hybrid were mapped by transcriptome profiling to the QTL regions as possible candidate genes. In parallel, a major QTL for yield heterosis, rice heterosis 8 (RH8), was found to be the DTH8/Ghd8/LHD1 gene. Based on the shared allelic heterozygosity of RH8 in many hybrid rice cultivars, a common mechanism for yield heterosis in the present commercial hybrid rice is proposed.

Excavation of Pid3 orthologs with differential resistance spectra to Magnaporthe oryzae in rice resource.

Twenty-six orthologs of the rice blast resistance gene Pid3 from cultivated varieties and wild rice accessions distributed in different areas were cloned by allele mining. Sequence analysis showed that while each of the orthologous genes from indica varieties and most wild accessions encodes a complete NBS-LRR protein, each of the proteins encoded by those from japonica varieties and few wild rice accessions presents a premature termination. Eleven of the 26 orthologs were selected for blast resistance testing by transforming into the blast susceptible rice variety TP309, respectively. Inoculation of 23 M. oryzae strains collected from diverse regions of China to the respective transgenic plants revealed that 6 Pid3 orthologs showed susceptible to all the tested strains, while the other 5 orthologs showed differential resistance spectra in a gradually spectrum-widen order as Pid3-W3, Pid3-W4, Pid3-I3, Pid3-W5 and Pid3-I1. Amino acid sequences alignment of these orthologs indicated that the sequence diversities between the blast resistance orthologs were mostly located in the LRR domain such as the substitutions of Q694H,D856H,Q896R,D899E etc. However, the differences between the resistance orthologs and the susceptible ones were mostly located in the NBS domain. The present experiments provide an example of that the ortholog evaluation of plant R genes could be an efficient way to expand the rice blast resistance and some other plant disease resistance as well for breeding.

Four receptor‐like cytoplasmic kinases regulate development and immunity in rice

Receptor-like cytoplasmic kinases (RLCKs) represent a large family of proteins in plants. However, few RLCKs have been well characterized. Here, we report the functional characterization of four rice RLCKs - OsRLCK57, OsRLCK107, OsRLCK118 and OsRLCK176 from subfamily VII. These OsRLCKs interact with the rice brassinosteroid receptor, OsBRI1 in yeast cell, but not the XA21 immune receptor. Transgenic lines silenced for each of these genes have enlarged leaf angles and are hypersensitive to brassinolide treatment compared to wild type rice. Transgenic plants silenced for OsRLCK57 had significantly fewer tillers and reduced panicle secondary branching, and lines silenced for OsRLCK107 and OsRLCK118 produce fewer seeds. Silencing of these genes decreased Xa21 gene expression and compromised XA21-mediated immunity to Xanthomonas oryzae pv. oryzae. Our study demonstrates that these OsRLCKs negatively regulate BR signalling, while positively regulating immune responses by contributing to the expression of the immune receptor XA21.

Characterization and fine mapping of a light-dependent leaf lesion mimic mutant 1 in rice.

Plants that spontaneously produce lesion mimics or spots, without any signs of obvious adversity, such as pesticide and mechanical damage, or pathogen infection, are so-called lesion mimic mutants (lmms). In rice, many lmms exhibit enhanced resistance to pathogens, which provides a unique opportunity to uncover the molecular mechanism underlying lmms. We isolated a rice light-dependent leaf lesion mimic mutant 1 (llm1). Lesion spots appeared in the leaves of the llm1 mutant at the tillering stage. Furthermore, the mutant llm1 had similar agronomic traits to wild type rice. Trypan blue and diamiobenzidine staining analyses revealed that the lesion spot formation on the llm1 mutant was due to programmed cell death and reactive oxygen species. The chloroplasts were severely damaged in the llm1 mutant, suggesting that chloroplast damage was associated with the formation of lesion spots in llm1. More importantly, llm1 exhibited enhanced resistance to bacterial blight pathogens within increased expression of pathogenesis related genes (PRs). Using a map-based cloning approach, we delimited the LLM1 locus to a 121-kb interval between two simple sequence repeat markers, RM17470 and RM17473, on chromosome 4. We sequenced the candidate genes on the interval and found that a base mutation had substituted adenine phosphate for thymine in the last exon of LOC_Os04g52130, which led to an amino acid change (Asp(388) to Val) in the llm1 mutant. Our investigation showed that the putative coproporphyrinogen III oxidase (CPOX) encoded by LOC_Os04g52130 was produced by LLM1 and that amino acid Asp(388) was essential for CPOX function. Our study provides the basis for further investigations into the mechanism underlying lesion mimic initiation associated with LLM1.

The durably resistant rice cultivar Digu activates defence gene expression before the full maturation of Magnaporthe oryzae appressorium.

Summary Rice blast caused by the fungal pathogen M agnaporthe oryzae is one of the most destructive diseases worldwide. Although the rice–M . oryzae interaction has been studied extensively, the early molecular events that occur in rice before full maturation of the appressorium during M . oryzae invasion are unknown. Here, we report a comparative transcriptomics analysis of the durably resistant rice variety Digu and the susceptible rice variety Lijiangxintuanheigu (LTH) in response to infection by M . oryzae (5, 10 and 20 h post‐inoculation, prior to full development of the appressorium). We found that the transcriptional responses differed significantly between these two rice varieties. Gene ontology and pathway analyses revealed that many biological processes, including extracellular recognition and biosynthesis of antioxidants, terpenes and hormones, were specifically activated in Digu shortly after infection. Forty‐eight genes encoding receptor kinases (RKs) were significantly differentially regulated by M . oryzae infection in Digu. One of these genes, LOC _Os08g10300, encoding a leucine‐rich repeat RK from the LRR VIII‐2 subfamily, conferred enhanced resistance to M . oryzae when overexpressed in rice. Our study reveals that a multitude of molecular events occur in the durably resistant rice Digu before the full maturation of the appressorium after M . oryzae infection and that membrane‐associated RKs play important roles in the early response.

The Multivesicular Bodies (MVBs)-Localized AAA ATPase LRD6-6 Inhibits Immunity and Cell Death Likely through Regulating MVBs-Mediated Vesicular Trafficking in Rice

Previous studies have shown that multivesicular bodies (MVBs)/endosomes-mediated vesicular trafficking may play key roles in plant immunity and cell death. However, the molecular regulation is poorly understood in rice. Here we report the identification and characterization of a MVBs-localized AAA ATPase LRD6-6 in rice. Disruption of LRD6-6 leads to enhanced immunity and cell death in rice. The ATPase activity and homo-dimerization of LRD6-6 is essential for its regulation on plant immunity and cell death. An ATPase inactive mutation (LRD6-6E315Q) leads to dominant-negative inhibition in plants. The LRD6-6 protein co-localizes with the MVBs marker protein RabF1/ARA6 and interacts with ESCRT-III components OsSNF7 and OsVPS2. Further analysis reveals that LRD6-6 is required for MVBs-mediated vesicular trafficking and inhibits the biosynthesis of antimicrobial compounds. Collectively, our study shows that the AAA ATPase LRD6-6 inhibits plant immunity and cell death most likely through modulating MVBs-mediated vesicular trafficking in rice.

Disruption of OsEXO70A1 Causes Irregular Vascular Bundles and Perturbs Mineral Nutrient Assimilation in Rice

Normal uptake, transportation, and assimilation of primary nutrients are essential to plant growth. Tracheary elements (TEs) are tissues responsible for the transport of water and minerals and characterized by patterned secondary cell wall (SCW) thickening. Exocysts are involved in the regulation of SCW deposition by mediating the targeted transport of materials and enzymes to specific membrane areas. EXO70s are highly duplicated in plants and provide exocysts with functional specificity. In this study, we report the isolation of a rice mutant rapid leaf senescence2 (rls2) that exhibits dwarfism, ferruginous spotted necrotic leaves, decreased hydraulic transport, and disordered primary nutrient assimilation. Histological analysis of rls2-1 mutants has indicated impaired cell expansion, collapsed vascular tissues, and irregular SCW deposition. Map-based cloning has revealed that RLS2 encodes OsEXO70A1, which is one of the 47 members of EXO70s in rice. RLS2 was widely expressed and spatially restricted in vascular bundles. Subcellular localization analysis demonstrated that RLS2 was present on both membrane and nuclear regions. Expression analysis revealed that mutations in rls2 triggers transcriptional fluctuation of orthologous EXO70 genes and affects genes involved in primary nutrient absorption and transport. In brief, our study revealed that RLS2 is required for normal vascular bundle differentiation and primary nutrient assimilation.

Overexpression of OsDof12 affects plant architecture in rice (Oryza sativa L.)

Dof (DNA binding with one finger) proteins, a class of plant-specific transcription factors, are involved in plant growth and developmental processes and stress responses. However, their biological functions remain to be elucidated, especially in rice (Oryza sativa L.). Previously, we have reported that OsDof12 can promote rice flowering under long-day conditions. Here, we further investigated the other important agronomical traits of the transgenic plants overexpressing OsDof12 and found that overexpressing OsDof12 could lead to reduced plant height, erected leaf, shortened leaf blade, and smaller panicle resulted from decreased primary and secondary branches number. These results implied that OsDof12 is involved in rice plant architecture formation. Furthermore, we performed a series of Brassinosteroid (BR)-responsive tests and found that overexpression of OsDof12 could also result in BR hyposensitivity. Of note, in WT plants the expression of OsDof12 was found up-regulated by BR treatment while in OsDof12 overexpression plants two positive BR signaling regulators, OsBRI1 and OsBZR1, were significantly down-regulated, indicating that OsDof12 may act as a negative BR regulator in rice. Taken together, our results suggested that overexpression of OsDof12 could lead to altered plant architecture by suppressing BR signaling. Thus, OsDof12 might be used as a new potential genetic regulator for future rice molecular breeding.