Jiandong Bao

Specific adaptation of Ustilaginoidea virens in occupying host florets revealed by comparative and functional genomics

Ustilaginoidea virens (Cooke) Takah is an ascomycetous fungus that causes rice false smut, a devastating emerging disease worldwide. Here we report a 39.4 Mb draft genome sequence of U. virens that encodes 8,426 predicted genes. The genome has ~25% repetitive sequences that have been affected by repeat-induced point mutations. Evolutionarily, U. virens is close to the entomopathogenic Metarhizium spp., suggesting potential host jumping across kingdoms. U. virens possesses reduced gene inventories for polysaccharide degradation, nutrient uptake and secondary metabolism, which may result from adaptations to the specific floret infection and biotrophic lifestyles. Consistent with their potential roles in pathogenicity, genes for secreted proteins and secondary metabolism and the pathogen-host interaction database genes are highly enriched in the transcriptome during early infection. We further show that 18 candidate effectors can suppress plant hypersensitive responses. Together, our analyses offer new insights into molecular mechanisms of evolution, biotrophy and pathogenesis of U. virens.

Comparative genomics identifies the Magnaporthe oryzae avirulence effector AvrPi9 that triggers Pi9‐mediated blast resistance in rice

We identified the Magnaporthe oryzae avirulence effector AvrPi9 cognate to rice blast resistance gene Pi9 by comparative genomics of requisite strains derived from a sequential planting method. AvrPi9 encodes a small secreted protein that appears to localize in the biotrophic interfacial complex and is translocated to the host cell during rice infection. AvrPi9 forms a tandem gene array with its paralogue proximal to centromeric region of chromosome 7. AvrPi9 is expressed highly at early stages during initiation of blast disease. Virulent isolate strains contain Mg-SINE within the AvrPi9 coding sequence. Loss of AvrPi9 did not lead to any discernible defects during growth or pathogenesis in M. oryzae. This study reiterates the role of diverse transposable elements as off-switch agents in acquisition of gain-of-virulence in the rice blast fungus. The prevalence of AvrPi9 correlates well with the avirulence pathotype in diverse blast isolates from the Philippines and China, thus supporting the broad-spectrum resistance conferred by Pi9 in different rice growing areas. Our results revealed that Pi9 and Piz-t at the Pi2/9 locus activate race specific resistance by recognizing sequence-unrelated AvrPi9 and AvrPiz-t genes, respectively.

Molecular evolution and selection of a gene encoding two tandem microRNAs in rice

It has been shown that overexpression of MIR156b/c resulted in a bushy phenotype in maize and rice. Our results indicated that the MIR156b/c locus was highly conserved among cereals, but not in dicots and that genome duplication events played an important role in the evolution of the miR156 family. Genetic diversity investigation at the locus indicated that only ∼9% of nucleotide diversity observed in wild rice (O. rufigogon) was maintained in the cultivated rice and the neutral model was rejected (P < 0.05) based on Tajimas D and Fu and Lis D ∗ and F ∗ tests. To our knowledge, this is the first example of miRNA gene to be targeted by both natural and domestication selection in plants.

Evidence of selectively driven codon usage in rice: Implications for GC content evolution of Gramineae genes

Two gene classes characterized by high and low GC content have been found in rice and other cereals, but not dicot genomes. We used paralogs with high and low GC contents in rice and found: (a) a greater increase in GC content at exonic fourfold‐redundant sites than at flanking introns; (b) with reference to their orthologs in Arabidopsis, most substitution sites between the two kinds of paralogs are found at 2‐ and 4‐degenerate sites with a T → C mode, while A → C and A → G play major roles at 0‐degenerate sites; and (c) high‐GC genes have greater bias and codon usage is skewed toward codons that are preferred in highly expressed genes. We believe this is strong evidence for selectively driven codon usage in rice. Another cereal, maize, also showed the same trend as in rice. This represents a potential evolutionary process for the origin of genes with a high GC content in rice and other cereals.

Post-domestication selection in the maize starch pathway.

Modern crops have usually experienced domestication selection and subsequent genetic improvement (post-domestication selection). Chinese waxy maize, which originated from non-glutinous domesticated maize (Zea mays ssp. mays), provides a unique model for investigating the post-domestication selection of maize. In this study, the genetic diversity of six key genes in the starch pathway was investigated in a glutinous population that included 55 Chinese waxy accessions, and a selective bottleneck that resulted in apparent reductions in diversity in Chinese waxy maize was observed. Significant positive selection in waxy (wx) but not amylose extender1 (ae1) was detected in the glutinous population, in complete contrast to the findings in non-glutinous maize, which indicated a shift in the selection target from ae1 to wx during the improvement of Chinese waxy maize. Our results suggest that an agronomic trait can be quickly improved into a target trait with changes in the selection target among genes in a crop pathway.

A host plant genome (Zizania latifolia) after a century-long endophyte infection

Despite the importance of host-microbe interactions in natural ecosystems, agriculture and medicine, the impact of long-term (especially decades or longer) microbial colonization on the dynamics of host genomes is not well understood. The vegetable crop Jiaobai with enlarged edible stems was domesticated from wild Zizania latifolia (Oryzeae) approximately 2000xa0years ago as a result of persistent infection by a fungal endophyte, Ustilago esculenta. Asexual propagation via infected rhizomes is the only means of Jiaobai production, and the Z.xa0latifolia-endophyte complex has been maintained continuously for two centuries. Here, genomic analysis revealed that cultivated Z.xa0latifolia has a significantly smaller repertoire of immune receptors compared with wild Z.xa0latifolia. There are widespread gene losses/mutations and expression changes in the plant-pathogen interaction pathway in Jiaobai. These results show that continuous long-standing endophyte association can have a major effect on the evolution of the structural and transcriptomic components of the host genome.

Genome-Wide Identification of New Reference Genes for qRT-PCR Normalization under High Temperature Stress in Rice Endosperm

qRT-PCR is one of the most popular approaches to analyze specific gene expression level, and stably expressed reference genes are essential to obtain reliable results. However, many reference genes are only stable under certain circumstances and different reference genes might be required in different experiments. High temperature is a common stress that affects rice endosperm development and it has become a hot topic recently. Although study about reference genes at different developmental stages in rice has been reported, these genes may not be suitable to study high temperature mediated responses especially in endosperm. In our quest for proper reference genes to quantify gene expression in rice endosperm under high temperature, we studied 6 candidate genes selected from the transcriptome data and 11 housekeeping genes. All genes were analyzed with qRT-PCR and the expression stability was assessed with software geNorm and NormFinder. Fb15 and eIF-4a were identified as the two most stable genes in endosperm at different developmental stages, while high temperature treatment has a least effect on expression of Fb15 and UBQ5 in rice endosperm. Our results provide some good candidate reference genes for qRT-PCR normalization in rice endosperm under different temperatures.

Directional Selection from Host Plants Is a Major Force Driving Host Specificity in Magnaporthe Species

One major threat to global food security that requires immediate attention, is the increasing incidence of host shift and host expansion in growing number of pathogenic fungi and emergence of new pathogens. The threat is more alarming because, yield quality and quantity improvement efforts are encouraging the cultivation of uniform plants with low genetic diversity that are increasingly susceptible to emerging pathogens. However, the influence of host genome differentiation on pathogen genome differentiation and its contribution to emergence and adaptability is still obscure. Here, we compared genome sequence of 6 isolates of Magnaporthe species obtained from three different host plants. We demonstrated the evolutionary relationship between Magnaporthe species and the influence of host differentiation on pathogens. Phylogenetic analysis showed that evolution of pathogen directly corresponds with host divergence, suggesting that host-pathogen interaction has led to co-evolution. Furthermore, we identified an asymmetric selection pressure on Magnaporthe species. Oryza sativa-infecting isolates showed higher directional selection from host and subsequently tends to lower the genetic diversity in its genome. We concluded that, frequent gene loss or gain, new transposon acquisition and sequence divergence are host adaptability mechanisms for Magnaporthe species, and this coevolution processes is greatly driven by directional selection from host plants.

Identification of glutinous maize landraces and inbred lines with altered transcription of waxy gene

Waxy maize has little or very low content of amylose (<5xa0%) in grain starch and carries null mutations of the waxy (Wx) gene. With important uses as fresh maize or as an ingredient in food, textile, adhesive, and paper industries, two types of wx allele, wx-D7 and wx-D10, had been identified. In the present study, 10 accessions carrying neither wx-D7 nor wx-D10 allele were identified from a collection of 325 waxy maize accessions. The Wx locus of these 10 accessions was sequenced, but no potential causative mutation was detected. Further sequencing of the full-length complementary DNAs revealed that 6 of the 10 accessions had alternative splicing patterns while the other 4 had wild-type Wx transcripts. Among the six accessions, one generated transcripts identical to the wx-D7 allele, two had the same transcripts as the wx-D10 allele, another two generated transcripts with deletion of the 10th and the 11th exon, yet the other one produced transcripts of various lengths due to deletions from part of the 2nd exon to part of the 12th exon. The wx alleles with the above alternative splicing modes are referred to as wx-tD7, wx-tD10, wx-tD10-11, and wx-tD2-12, respectively. Real-time quantitative reverse-transcription polymerase chain reaction analysis of two waxy accessions that produced Wx transcripts showed significantly decreased expression, having only 47.3 and 3.6xa0% transcription level compared with B73. Sequence analysis of deletions in the transcripts with comparison with wild one showed short direct repeats at deletion endpoints, similar to reported signatures of DNA deletions. The waxy accessions present unique wx alleles for waxy maize breeding as well as for transcriptional regulation studies in plants.

Population genomic analysis of the rice blast fungus reveals specific events associated with expansion of three main clades

We examined the genomes of 100 isolates of Magnaporthe oryzae (Pyricularia oryzae), the causal agent of rice blast disease. We grouped current field populations of M. oryzae into three major globally distributed groups. A genetically diverse group, clade 1, which may represent a group of closely related lineages, contains isolates of both mating types. Two well-separated clades, clades 2 and 3, appear to have arisen as clonal lineages distinct from the genetically diverse clade. Examination of genes involved in mating pathways identified clade-specific diversification of several genes with orthologs involved in mating behavior in other fungi. All isolates within each clonal lineage are of the same mating type. Clade 2 is distinguished by a unique deletion allele of a gene encoding a small cysteine-rich protein that we determined to be a virulence factor. Clade 3 isolates have a small deletion within the MFA2 pheromone precursor gene, and this allele is shared with an unusual group of isolates we placed within clade 1 that contain AVR1-CO39 alleles. These markers could be used for rapid screening of isolates and suggest specific events in evolution that shaped these populations. Our findings are consistent with the view that M. oryzae populations in Asia generate diversity through recombination and may have served as the source of the clades 2 and 3 isolates that comprise a large fraction of the global population.