Current understanding on Villosiclava virens,a unique flower‐infecting fungus causing rice false smut disease

Villosiclava virens (Vv) is an ascomycete fungal pathogen that causes false smut disease in rice. Recent reports have revealed some interesting aspects of the enigmatic pathogen to address the question of why it specifically infects rice flowers and converts a grain into a false smut ball. Comparative and functional genomics have suggested specific adaptation of Vv in the colonization of rice flowers. Anatomical studies have disclosed that Vv specifically infects rice stamen filaments before heading and intercepts seed formation. In addition, Vv can occupy the whole inner space of a spikelet embracing all floral organs and activate the rice grain‐filling network, presumably for nutrient acquisition to support the development of the false smut ball. This profile provides a general overview of the rice false smut pathogen, and summarizes advances in the Vv life cycle, genomics and genetics, and the molecular Vv–rice interaction. Current understandings of the Vv–rice pathosystem indicate that it is a unique and interesting system which can enrich the study of plant–pathogen interactions. Taxonomy: Ustilaginoidea virens is the anamorph form of the pathogen (Kingdom Fungi; Phylum Ascomycota; Class Ascomycetes; Subclass Incertae sedis; Order Incertae sedis; Family Incertae sedis; Genus Ustilaginoidea). The teleomorph form is Villosiclava virens (Kingdom Fungi; Phylum Ascomycota; Class Ascomycetes; Subclass Sordariomycetes; Order Hypocreales; Family Clavicipitaceae; Genus Villosiclava). Disease symptoms: The only visible symptom is the replacement of rice grains by ball‐shaped fungal mycelia, namely false smut balls. When maturing, the false smut ball is covered with powdery chlamydospores, and the colour changes to yellowish, yellowish orange, green, olive green and, finally, to greenish black. Sclerotia are often formed on the false smut balls in autumn. Identification and detection: Vv conidia are round to elliptical, measuring 3–5 μm in diameter. Chlamydospores are ornamented with prominent irregularly curved spines, which are 200–500 nm in length. The sclerotia are black, horseshoe‐shaped and irregular oblong or flat, ranging from 2 to 20 mm. Nested polymerase chain reaction (PCR) and quantitative PCR have been developed to specifically detect Vv presence in rice tissues and other biotic and abiotic samples in fields. Host range: Rice is the primary host for Vv. Natural infection by Vv has been found on several paddy field weeds, including Digitaria marginata, Panicum trypheron, Echinochloa crusgalli and Imperata cylindrica. However, the occurrence of infection in these potential alternative hosts is very rare. Life cycle: Vv infects rice spikelets at the late rice booting stage, and produces false smut balls covered with dark‐green chlamydospores. Occasionally, sclerotia form on the surface of false smut balls in late autumn when the temperature fluctuates greatly between day and night. Both chlamydospores and sclerotia may serve as primary infection sources. Rainfall at the rice booting stage is a major environmental factor resulting in epidemics of rice false smut disease. Disease control: The use of fungicides is the major approach for the control of Vv. Several fungicides, such as cuproxat SC, copper oxychloride, tebuconazole, propiconazole, difenoconazole and validamycin, are often applied. However, the employment of resistant rice cultivars and genes has been limited, because of the poor understanding of rice resistance to Vv. Useful websites: Villosiclava virens genome sequence: http://www.ncbi.nlm.nih.gov/Traces/wgs/?val=JHTR01#contigs     

稻绿核菌(稻曲病菌)分离方法的比较研究

作者对不同情况下稻曲病菌的分离方法进行了比较研究。结果表明成熟早期稻曲球上的绝大多数新鲜的厚垣孢子具有萌发能力,及时进行分离培养是病原菌成功分离的关键。随保存时间的延长,厚垣孢子萌发能力急剧下降;消毒处理可杀死大部分的厚垣孢子。菌核可长期保存并保持极高的萌发生长能力,是稻曲病菌分离最为理想的材料。稻曲球中部的致密菌丝组织分离难度较大,只能作为稻曲病菌分离的一种补救方法。     

Suppression of α‐amylase genes improves quality of rice grain ripened under high temperature

High temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism‐related genes. Here we report the involvement of a starch‐hydrolyzing enzyme, α‐amylase, in high temperature‐triggered grain chalkiness. In developing seeds, high temperature induced the expression of α‐amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as α‐amylase activity, while it decreased an α‐amylase‐repressing plant hormone, ABA, suggesting starch to be degraded by α‐amylase in developing grains under elevated temperature. Furthermore, RNAi‐mediated suppression of α‐amylase genes in ripening seeds resulted in fewer chalky grains under high‐temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of α‐amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.     

Physiological characteristics and related gene expression of after‐ripening on seed dormancy release in rice

After‐ripening is a common method used for dormancy release in rice. In this study, the rice variety Jiucaiqing (Oryza sativa L. subsp. japonica) was used to determine dormancy release following different after‐ripening times (1, 2 and 3 months). Germination speed, germination percentage and seedling emergence increased with after‐ripening; more than 95% germination and 85% seedling emergence were observed following 1 month of after‐ripening within 10 days of imbibition, compared with <45% germination and 20% seedling emergence in freshly harvested seed. Hence, 3 months of after‐ripening could be considered a suitable treatment period for rice dormancy release. Dormancy release by after‐ripening is mainly correlated with a rapid decline in ABA content and increase in IAA content during imbibition. Subsequently, GA₁/ABA, GA₇/ABA, GA₁₂/ABA, GA₂₀/ABA and IAA/ABA ratios significantly increased, while GA₃/ABA, GA₄/ABA and GAs/IAA ratio significantly decreased in imbibed seeds following 3 months of after‐ripening, thereby altering α‐amylase activity during seed germination. Peak α‐amylase activity occurred at an earlier germination stage in after‐ripened seeds than in freshly harvested seeds. Expression of ABA, GA and IAA metabolism genes and dormancy‐related genes was regulated by after‐ripening time upon imbibition. Expression of OsCYP707A5, OsGA2ox1, OsGA2ox2, OsGA2ox3, OsILR1, OsGH3‐2, qLTG3‐1 and OsVP1 increased, while expression of Sdr4 decreased in imbibed seeds following 3 months of after‐ripening. Dormancy release through after‐ripening might be involved in weakening tissues covering the embryo via qLTG3‐1 and decreased ABA signalling and sensitivity via Sdr4 and OsVP1.     

The 14‐3‐3 protein GF14f negatively affects grain filling of inferior spikelets of rice (Oryza sativa L.)

In rice (Oryza sativa L.), later flowering inferior spikelets (IS), which are located on proximal secondary branches, fill slowly and produce smaller and lighter grains than earlier flowering superior spikelets (SS). Many genes have been reported to be involved in poor grain filling of IS, however the underlying molecular mechanisms remain unclear. The present study determined that GF14f, a member of the 14‐3‐3 protein family, showed temporal and spatial differences in expression patterns between SS and IS. Using GF14f–RNAi plants, we observed that a reduction in GF14f expression in the endosperm resulted in a significant increase in both grain length and weight, which in turn improved grain yield. Furthermore, pull‐down assays indicated that GF14f interacts with enzymes that are involved in sucrose breakdown, starch synthesis, tricarboxylic acid (TCA) cycle and glycolysis. At the same time, an increase in the activity of sucrose synthase (SuSase), adenosine diphosphate‐glucose pyrophosphorylase (AGPase), and starch synthase (StSase) was observed in the GF14f–RNAi grains. Comprehensive analysis of the proteome and metabolite profiling revealed that the abundance of proteins related to the TCA cycle, and glycolysis increased in the GF14f–RNAi grains together with several carbohydrate intermediates. These results suggested that GF14f negatively affected grain development and filling, and the observed higher abundance of the GF14f protein in IS compared with SS may be responsible for poor IS grain filling. The study provides insights into the molecular mechanisms underlying poor grain filling of IS and suggests that GF14f could serve as a potential tool for improving rice grain filling.     

ChIP‐Seq of ERα and RNA polymerase II defines genes differentially responding to ligands

We used ChIP‐Seq to map ERα‐binding sites and to profile changes in RNA polymerase II (RNAPII) occupancy in MCF‐7 cells in response to estradiol (E2), tamoxifen or fulvestrant. We identify 10 205 high confidence ERα‐binding sites in response to E2 of which 68% contain an estrogen response element (ERE) and only 7% contain a FOXA1 motif. Remarkably, 596 genes change significantly in RNAPII occupancy (59% up and 41% down) already after 1 h of E2 exposure. Although promoter proximal enrichment of RNAPII (PPEP) occurs frequently in MCF‐7 cells (17%), it is only observed on a minority of E2‐regulated genes (4%). Tamoxifen and fulvestrant partially reduce ERα DNA binding and prevent RNAPII loading on the promoter and coding body on E2‐upregulated genes. Both ligands act differently on E2‐downregulated genes: tamoxifen acts as an agonist thus downregulating these genes, whereas fulvestrant antagonizes E2‐induced repression and often increases RNAPII occupancy. Furthermore, our data identify genes preferentially regulated by tamoxifen but not by E2 or fulvestrant. Thus (partial) antagonist loaded ERα acts mechanistically different on E2‐activated and E2‐repressed genes.     

Putative zeatin O‐glucosyltransferase OscZOG1 regulates root and shoot development and formation of agronomic traits in rice

As a ubiquitous reaction, glucosylation controls the bioactivity of cytokinins in plant growth and development.Here we show that genetic manipulation of zeatin-Oglucosylation regulates the formation of important agronomic traits in rice by manipulating the expression of OscZOG1 gene,encoding a putative zeatin O-glucosyltransferase. We found that OscZOG1 was preferentially expressed in shoot and root meristematic tissues and nascent organs. The growth of lateral roots was stimulated in the overexpression lines, but inhibited in RNA interference lines. In shoots, knockdown of OscZOG1 expression by RNA interference significantly improved tillering, panicle branching, grain number per panicle and seed size, which are important agronomic traits for grain yield. In contrast, constitutive expression of OscZOG1 leads to negative effects on the formation of the grain-yielding traits with a marked increase in the accumulation levels of cis-zeatin O-glucoside(c ZOG) in the transgenic rice plants. In this study,our findings demonstrate the feasibility of improving the critical yield-determinant agronomic traits, including tiller number, panicle branches, total grain number per panicle and grain weight by downregulating the expression level of OscZOG1. Our results suggest that modulating the levels of cytokinin glucosylation can function as a fine-tuning switch in regulating the formation of agronomic traits in rice.     

Discovery of rice essential genes by characterizing a CRISPR‐edited mutation of closely related rice MAP kinase genes

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co‐expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA‐gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen‐activated protein kinase (MPK) genes in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR‐edited mutants. The true knock‐out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR‐induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely related MPK genes, we demonstrated 45–86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene‐free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy.     

FLOURY ENDOSPERM11‐2 encodes plastid HSP70‐2 involved with the temperature‐dependent chalkiness of rice (Oryza sativa L.) grains

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature‐sensitive floury endosperm11‐2 (flo11‐2) mutant was isolated from ion beam‐irradiated rice of 1116 lines. The flo11‐2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole‐exome sequencing of the flo11‐2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid‐localized 70‐kDa heat shock protein 2 (cpHSP70‐2). The cpHSP70‐2 of the flo11‐2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11‐2 mutants that express the wild‐type cpHSP70‐2 showed significantly lower chalkiness than the flo11‐2 mutant. Moreover, the accumulation level of cpHSP70‐2 was negatively correlated with the chalky ratio, indicating that cpHSP70‐2 is a causal gene for the chalkiness of the flo11‐2 mutant. The intrinsic ATPase activity of recombinant cpHSP70‐2 was lower by 23% at V_max for the flo11‐2 mutant than for the wild type. The growth of DnaK‐defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild‐type DnaK. The results indicate that the lowered cpHSP70‐2 function is involved with the chalkiness of the flo11‐2 mutant.     

Three‐dimensional genetic networks among seed oil‐related traits,metabolites and genes reveal the genetic foundations of oil synthesis in soybean

Although the biochemical and genetic basis of lipid metabolism is clear in Arabidopsis, there is limited information concerning the relevant genes in Glycine max (soybean). To address this issue, we constructed three‐dimensional genetic networks using six seed oil‐related traits, 52 lipid metabolism‐related metabolites and 54 294 SNPs in 286 soybean accessions in total. As a result, 284 and 279 candidate genes were found to be significantly associated with seed oil‐related traits and metabolites by phenotypic and metabolic genome‐wide association studies and multi‐omics analyses, respectively. Using minimax concave penalty (MCP) and smoothly clipped absolute deviation (SCAD) analyses, six seed oil‐related traits were found to be significantly related to 31 metabolites. Among the above candidate genes, 36 genes were found to be associated with oil synthesis (27 genes), amino acid synthesis (four genes) and the tricarboxylic acid (TCA) cycle (five genes), and four genes (GmFATB1a, GmPDAT, GmPLDα1 and GmDAGAT1) are already known to be related to oil synthesis. Using this information, 133 three‐dimensional genetic networks were constructed, 24 of which are known, e.g. pyruvate–GmPDAT–GmFATA2–oil content. Using these networks, GmPDAT, GmAGT and GmACP4 reveal the genetic relationships between pyruvate and the three major nutrients, and GmPDAT, GmZF351 and GmPgs1 reveal the genetic relationships between amino acids and seed oil content. In addition, GmCds1, along with average temperature in July and the rainfall from June to September, influence seed oil content across years. This study provides a new approach for the construction of three‐dimensional genetic networks and reveals new information for soybean seed oil improvement and the identification of gene function.     

Genome‐wide analysis of polymorphisms identified domestication‐associated long low‐diversity region carrying important rice grain size/weight quantitative trait loci

Rice grain size and weight are major determinants of grain quality and yield and so have been under rigorous selection since domestication. However, the genetic basis for contrasting grain size/weight trait among Indian germplasms and their association with domestication‐driven evolution is not well understood. In this study, two long (LGG) and two short grain (SGG) genotypes were resequenced. LGG (LGR and PB 1121) differentiated from SGG (Sonasal and Bindli) by 504 439 single nucleotide polymorphisms (SNPs) and 78 166 insertion‐and‐deletion polymorphisms. The LRK gene cluster was different and a truncation mutation in the LRK8 kinase domain was associated with LGG. Phylogeny with 3000 diverse rice accessions revealed that the four sequenced genotypes belonged to the japonica group and were at the edge of the clades indicating them to be the potential source of genetic diversity available in Indian rice germplasm. Six SNPs were significantly associated with grain size/weight and the top four of these could be validated in mapping a population, suggesting this study as a valuable resource for high‐throughput genotyping. A contiguous long low‐diversity region (LDR) of approximately 6 Mb carrying a major grain weight quantitative trait loci (harbouring OsTOR gene) was identified on Chromosome 5. This LDR was identified as an evolutionary important site with significant positive selection and multiple selection sweeps, and showed association with many domestication‐related traits, including grain size/weight. The aus population retained more allelic variations in the LDR than the japonica and indica populations, suggesting it to be one of the divergence loci. All the data and analyses can be accessed from the RiceSzWtBase database.