Functional genomics in the rice blast fungus to unravel the fungal pathogenicity

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.     

臭氧水对草莓及拟盘多毛孢影响的研究

通过组织分离纯化和分子生物学鉴定,明确了引起草莓叶部新病的致病菌为拟盘多毛孢。以喷洒清水为空白对照,利用不同质量浓度的臭氧水直接喷洒致病菌和生长期的草莓植株,研究臭氧水对致病菌和草莓植株的浓度效应,结果显示：低浓度臭氧水（0.5~0.8 mg·L^-1）对草莓植株的生理生态变化和致病菌的生长影响较小;中浓度臭氧水（2.2~2.5 mg·L^-1）可以显著抑制致病菌的生长,并促进草莓植株的生长;高浓度臭氧水（4.0~4.3 mg·L^-1）可以很好地抑制致病菌的生长,但对草莓叶片有较严重的腐蚀作用。因此,中浓度（2.2~2.5 mg·L^-1）是喷洒草莓的最适臭氧水浓度。     

悬崖与非悬崖生境下中华山蓼克隆整合能力及其表型差异比较

中华山蓼(Oxyria sinensis)是一种典型的根状茎型多年生草本,能在悬崖岩面上形成相当大的克隆。为了进一步了解中华山蓼在悬崖生境中的克隆生长特性及其对悬崖生境的适应对策,本研究比较了悬崖和非悬崖两种生境条件下生长的中华山蓼的克隆整合能力和形态可塑性,拟探讨如下问题:(1)中华山蓼是否具有克隆整合能力,如果有,其整合能力有多大?(2)中华山蓼在不同的生境条件下的克隆整合能力和形态上是否具有显著性差异?研究结果表明,中华山蓼具有克隆整合能力,但大多数的克隆其整合能力均不大。悬崖生境和非悬崖生境中的中华山蓼的整合能力之间没有显著性差异。中华山蓼在比叶面积、叶柄长、叶/叶柄生物量分配等性状上表现出了较强的可塑性,这些性状可能是中华山蓼对悬崖生境的适应对策之一。     

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.     

Genome-wide analysis of nucleotide-binding site disease resistance genes in Medicago truncatula

The class of nucleotide-binding site （NBS）- Leucine-rich repeat （LRR） disease resistance genes play an important role in defending plants from a variety of pathogens and insect pests. Consequently, many NBS-LRR genes have been identified in various plant species. In this study, we identified 617 NBS-encoding genes in the Medicago truncatula genome （Mt3.5v5） and divided them into two groups, regular （490） and non-regular （127） NBS- LRR genes. The regular NBS-LRR genes were character- ized on the bases of structural diversity, chromosomal location, gene duplication, conserved protein motifs, and EST expression profiling. According to N-terminal motifs and LRR motifs, the 490 regular NBS-LRR genes were then classified into 10 types： CC-NBS （4）, CC-NBS-LRR （212）, TIR-NBS （20）, TIR-NBS-LRR （160）, TIR-NBS-TIR （1）, TIR-NBS-TIR-LRR （2）, NBS-TIR （7）, NBS-TIR-LRR （1）, NBS （10）, and NBS-LRR （73）. Analysis of the phys- ical location and duplications of the regular NBS-LRR genes revealed that the M. truncatula genome is similar to rice. Interestingly, we found that TIR-type genes are more frequently expressed than non-TIR-type genes in M. trun- catula, whereas the number of non-TIR-type regular NBS- LRR genes was greater than TIR-type genes, suggesting the gene expression was not associated with the total number of NBS-LRR genes. Moreover, we found that the phylogenetic tree supported our division of the regular NBS-LRR genes into two distinct clades （TIR-type and non-TIR-type）, but some of the non-TIR-type lineages contain TIR-type genes. These analyses provide a robust database of NBS-LRR genes in M. truncatula that will facilitate the isolation of new resistance genes and breeding strategies to engineer disease resistance in leguminous crop     

Effects of inoculating fungi on agilawood formation in Aquilaria sinensis

Agilawood is a costly heartwood medicine obtained from Aquilaria sinensis with active ingredients mainly composed of volatile and semi-volatile substances. However, the formation time of agilawood is quite long and little is known about its formation mechanism. Two highly active fungi obtained from natural agilawood were inoculated on A. sinensis trees to understand their interaction processes and elucidate the transformation rules of induced chemical compositions within different test periods. The results demonstrated that the fungi could successfully colonize living tissues and cells and activate the host defense system, resulting in agilawood accumulation. With increasing time, the main components of A. sinensis converted into constituents or analogs of agilawood and the host exhibited "self-injury" to prevent fungal intrusion and protect other tissues. The data presented here could provide scientific basis for producing agilawood with the two new fungi in a safe, feasible, and sustainable manner without destroying rare Aquilaria plants.     

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

Bombyx mori bidensovirus: The type species of the new genus Bidensovirus in the new family Bidnaviridae

Bombyx mori bidensovirus （BmBDV）, which had been assigned to Densovirinae in Parvoviridae previously, replicates mainly in silkworm midgut columnar cells and causes the fatal flacheric disease. In contrast to parvovirus, this virus possesses two single-stranded DNA genome segments and encodes a putative protein-primed DNA polymerase. The accumulating evidence sug- gests that it has unique characteristics different to parvovirus and adopts its own mechanisms for replication. So far, little is known about the replication mechanisms of BmBDV. In this review, we focus on the pathology associated with this virus and the viral biology such as viral genome structure, viral genes, and viral replication and expression strategies.     

Identification and bioinformatics analysis of pseudogenes from whole genome sequence of Phaeodactylum tricornutum

Pseudogenes share sequence similarities with functional genes, but in general they have lost their protein-coding ability. The identification of pseudogenes is a very important step in genome annotation. Phaeodactylum tricornutum is a marine diatom that is rich in polyunsaturated fatty acids (PUFAs). The genome of P. tricornutum has been completely sequenced. To identify pseudogenes in P. tricornutum, we developed a pipeline to discover and characterize pseudogenes. We identified a total of 1654 ‘true’ processed pseudogenes, 714 duplicated pseudogenes and 4729 pseudogene fragments. The results of the bioinformatics analysis indicated that the genome sequence of P. tricornutum contained many pseudogenes and pseudogene fragments.     

Construction of Streptomyces lydicus A01 transformant with the chit33 gene from Trichoderma harzianum CECT2413 and its biocontrol effect on Fusaria

Streptomyces lydicus A01 resists many plant pathogens (including Fusarium spp.) by producing the antifungal agent natamycin, which binds to the ergosterol of fungal cell membranes and inhibits the growth of pathogens. Trichoderma harzianum CECT2413 is a widely-distributed soil fungus that antagonizes several plant fungal pathogens (including Fusarium spp.) by producing chi-tinase and degrading chitin, a major component of the fungal cell wall. This study attempted to enhance the biocontrol effect of S. lydicus A01 on Fusarium spp. by transforming the chitinase gene of Trichoderma. Chitinase and natamycin could act synergisti-cally on both the cell walls and cell membranes of pathogens. The 33-kD chitinase-encoding gene (chit33) was cloned and conju-gal-transformed from T. harzianum CECT2413 to S. lydicus A01, and then confirmed via polymerase chain reaction (PCR) assays. Subsequent analyses using the 3,5-dinitrosalicylic acid (DNS) method and ultraviolet spectrophotometry showed that compared with its wild type strain (WT), the S. lydicus A01 conjugal transformant (CT) with chit33 gene exhibited substantially higher chi-tinase activity and natamycin production. The resistance of S. lydicus A01-chit33 CT and WT to four Fusaria in crops and vegetables was tested via the cup-plate method. Compared with the WT, the conjugal transformant of S. lydicus A01 with chit33 gene from T. harzianum CECT2413 showed greatly increased biocontrol effect on fusarium disease. This study would be beneficial to the development of high-quality antifungal bio-agents for agricultural applications via the synergy between the previously non-existent and pre-existing functions achieved through heterogeneous gene transformation.     

A wound-induced small polypeptide gene family is upregulated in soybean nodules

Small peptides function as key signals in processes, such as plant cell differentiation, organ development and defenses to biotic stresses. A large number of small peptide precursor genes have been predicted from the analysis of the soybean (Glycine max) whole genome DNA sequence. However, most of these genes have unknown characteristics and functions. In this report, we systemically searched for the gene families of small peptide precursors that are up-regulated in soybean nitrogen-fixing root nodules. We found 212 genes (encoding peptides shorter than 150 amino acids) that were up-regulated, and among them, 79 genes belong to 38 multiple-gene families, but the other 133 genes are unique. Twenty-eight of 38 families are conserved in Arabidopsis, but the other 10 only exist in legumes. We also identified 16 out of the 38 members of the wound-induced polypeptide (WIP) gene family to be upregulated in nitrogen-fixing nodules. We further analyzed homologs of WIP genes in Medicago, Lotus, Arabidopsis and Oryza species and found that a few homologous genes from Medicago truncatula and Lotus japonicus were also upregulated in their nodules and some WIP genes were induced by specific fungal pathogens on soybean and rice. Structure prediction indicated that all WIP prepropeptides contain a conserved DUF3774 domain (including two hydrophobic regions) and most of them have an N-terminal signal sequence. Fluorescence microscopy analysis of two WIP prepropeptides fused to GFP revealed that these proteins are located on the plasma membrane of tobacco leaf cells. Interestingly, 34 soybean WIP genes are clustered onto three soybean chromosomes, different from known peptide gene families (such as CLE). Among them, 11 highly identical genes are aligned on the 6th chromosome, 12 on the 12th, and 11 on the 13th chromosomes. Most of WIP genes from the 12th chromosome share the highest identities with their homologs on the 13th chromosome, suggesting that ancestral WIP genes could have originated from the 13th chromosome, then spread onto the 12th chromosome by chromosome homologous recombination; the new WIP genes could have existed in multiple copies by gene duplication which then spread onto the 6th chromosome. In Arabidopsis and Oryza species, half of the WIP genes are also aligned on one chromosome and showed higher identity with those from the soybean 12th and 13th chromosomes, suggesting that WIP genes originated from one common ancestor.     

Insights on evolution of virulence and resistance from the whole-genome analysis of a predominant methicillin-resistantStaphylococcus aureus clone sequence type 239 in China

Earlier, we reported that ST239 was the 15-year predominant methicillin-resistant Staphylococcus aureus （MRSA） clone in China. In this study, MRSA strain CN79 belonging to ST239 and isolated from blood was used to determine the whole tive genomics analysis was genome sequence. Compara- done between MRSA CN79 and 25 sequenced S. aureus in the NCBI GenBank data- base. A total of 2,734 protein-encoding genes were iden- tified in the MRSA CN79 genome, which carries 11 antibiotic resistance genes and 65 virulence genes. Two prophages phiCN79A and phiNM3-1ike were found on the MRSA CN79 genome. MRSA CN79 carries 30 specific genes that are absent from the 25 sequenced S. aureus genomes. Most of them were prophage-related genes. Several antibiotic resistance genes, such as [3-1actamase and ABC-type multidrug transport system gene, were located on the genomic island vSal3. The antibiotic resis- tance genes, such as tet （M）, ermA1, and blaZ, were also located on different transposons. The virulence genes sea,map, hlb, and sak are located on phiNM3-1ike prophage and the exotoxin genes are carried on the genomic island vSaa. These results suggest that ST239 MRSA strains are widespread owing to horizontal acquisition of the mobile genetic elements harbored antibiotic resistance genes and virulence genes in response to environmental selective pressures, such as antibiotics and the human immune sys- tem during evolution.     

云南当归软腐病的危害性及病原鉴定

云归受软腐病危害严重,为了解生产受损情况并明确其病原,调查了田间受害情况并对病原进行鉴定.田间调查发病株率,采集病害标本,观察植株病害症状,实验室分离、镜检鉴定病原.该病造成当归根部软腐,调查田块发病面积100%,发病株率20%左右,造成至少10%的产量损失或近20%的经济损失.病原为线虫,其形态与腐烂茎线虫的重要鉴别特征相符,且形态测量数据在原定种的范围内.结果显示云南当归软腐病由腐烂茎线虫(Ditylenchus destructor)引起.     

The genome sequence of the rice blast fungus Magnaporthe grisea

Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.     

Evaluating the influences of measurement time and frequency on soil respiration in a semiarid temperate grassland

Soil respiration(Soil R)is one of the largest CO2fluxes from terrestrial ecosystems to the atmosphere.The largely seasonal and daily patterns of Soil R in semiarid grassland ecosystems indicate that measurement time and frequency would have significant influences on the assessment of seasonal soil carbon release.Based on a three-year continuous measurement of Soil R in a semiarid grassland,we found that the Soil R value measured at around 10:00 o’clock local time was the closest to its daily mean,while the value at 14:00 o’clock was found to be the highest daily rate.A measurement frequency higher than every 10 days was necessary for estimating the seasonal Soil R and its temperature sensitivity(Q10)reasonably.Our study would be useful as guidelines for manual Soil R measurements and model data selection in semiarid temperate grasslands.     

广西珍珠湾红树林区中华乌塘鳢的时空分布特征

为了解广西珍珠湾红树林区中华乌塘鳢的天然资源量及其时空变化动态,为红树林生态系统生物多样性的保护和修复提供基础数据,本研究在广西珍珠湾红树林区的内、中、外滩各设置一个圆形封闭定置围网,于2013年的4季度（1月、4月、7月、10月）各选择3 d投放被标志的中华乌塘鳢（Bostrychus sinensis,区别于野生鱼）至围网中,并在次日用诱捕网捕获,基于标志重捕法的原理评估野生中华乌塘鳢的天然资源量。结果显示,珍珠湾红树林区中华乌塘鳢的全年平均种群密度为（18.74±4.04） ind./hm²,平均生物量为（1.44±0.37） kg/hm²,其中1月的生物量显著高于10月,其余时间的种群密度和生物量均无显著差异;其种群密度和生物量在红树林中无显著空间差异。红树林为中华乌塘鳢提供繁育场,生境的破坏和过度捕捞导致中华乌塘鳢天然资源量下降,应加强监管、保护和相关研究,促进中华乌塘鳢资源的恢复。     

Molecular characterization of H3N2 and H4N6 subtypes avian influenza viruses isolated from mallards in Poyang Lake, China in 2010

Poyang Lake is the largest inland freshwater lake in China and contains many species of wild birds and waterfowls.We conducted a survey of avian influenza viruses in nine semi-artificial waterfowl farms in Poyang Lake during January to March of 2010.Out of 1036 cloacal swabs collected,three H3N2 and one H4N6 influenza viruses were isolated from healthy mallards.All the isolates were genetically and phylogenetically characterized.The analysis of putative HA cleavage sites showed that all the four isolates possessed the molecular characteristics(QTRGL for H3N2 viruses,PEKASR for H4N6 virus) of lowly pathogenic avian influenza(LPAI) virus.The phylogenetic analysis of the viral genomes showed that all four virus isolates clustered in the Eurasian clade of influenza viruses.The M gene of the viruses possessed the highest homology with highly pathogenic H5N1 influenza viruses.In addition,co-infection of H3N2 and H4N6 in the same farm was observed.And interestingly,we isolated two subtypes viruses(H3N2 and H4N6) and their progeny virus(H3N2) with evidence of genome reassortment from the same farm,in which the PB1 and PB2 gene segments of H4N6 replaced those of the H3N2 strain.The results of animal infection experiments showed that all the four isolated viruses were lowly pathogenic to chickens and not pathogenic to mice,which was consistent with the results of genetic analysis.     

Blast fungus-induction and developmental and tissue-specific expression of a rice P450<Emphasis Type="Italic">CYP72A</Emphasis> gene cluster

Cytochrome P450 gene superfamily is widely involved in diverse processes of plant development and environmental responses including defense response to pathogens.We previously isolated a rice cDNA fragment in a DD-PCR screening for blast fungus-induced genes. In the current study, we isolated a CYP72A gene cluster consisting of 7 P450 CYP72A genes (CYP72A17-23) with the conserved cDNA sequence through the public rice genome data. There are total 14 putative CYP72A members in the rice genome, with high diversity at N-terminal sequences while high homology at C-terminal sequences of those 14 putative proteins. We analyzed expression profiles of the cloned 7 CYP72A genes during pathogen infection and development. The results showed that expression of CYP72A18, 19, 22 and 23 was differentially regulated in the incompatible and compatible interactions between rice and blast fungus. Except CYP72A20, a pseudogene, other 6 CYP72A genes also exhibited temporal and spatial expression patterns, respectively.These findings provide fundamental data for rice P450 gene function analysis.