Survival strategy of the salt-tolerant lactic acid bacterium,Tetragenococcus halophilus,to counteract koji mold,Aspergillus oryzae,in soy sauce brewing

In soy sauce brewing, the results of the fermentation of lactic acid greatly affect the quality of soy sauce. The soy sauce moromi produced with Aspergillus oryzae RIB40 allows the growth of Tetragenococcus halophilus NBRC 12172 but not T. halophilus D10. We isolated and identified heptelidic acid (HA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), produced by A. oryzae RIB40 as the growth inhibitor of the salt-tolerant lactic acid bacteria. The growth inhibition of T. halophilus D10 by HA was suggested to be associated with the direct inhibition of GAPDH activity under high salt environment. The difference in the susceptibility to HA among various strains of T. halophilus was caused by the mutations in the gene encoding GAPDH.     

Self/nonself recognition in Tuber melanosporum is not mediated by a heterokaryon incompatibility system

Vegetative incompatibility is a widespread phenomenon in filamentous ascomycetes, which limits formation of viable heterokaryons. Whether this phenomenon plays a role in maintaining the homokaryotic state of the hyphae during the vegetative growth of Tuber spp. Gene expression, polymorphism analysis as well as targeted in vitro experiments allowed us to test whether a heterokaryon incompatibility (HI) system operates in Tuber melanosporum. HI is controlled by different genetic systems, often involving HET domain genes and their partners whose interaction can trigger a cell death reaction. Putative homologues to HI-related genes previously characterized in Neurospora crassa and Podospora anserina were identified in the T. melanosporum genome. However, only two HET domain genes were found. In many other ascomycetes HET domains have been found within different genes including some members of the NWD (NACHT and WD-repeat associated domains) gene family of P. anserina. More than 50 NWD homologues were found in T. melanosporum but none of these contain a HET domain. All these T. melanosporum paralogs showed a conserved gene organization similar to the microexon genes only recently characterized in Schistosoma mansoni. Expression data of the annotated HI-like genes along with low allelic polymorphism suggest that they have cellular functions unrelated to HI. Moreover, morphological analyses did not provide evidence for HI reactions between pairs of genetically different T. melanosporum strains. Thus, the maintenance of the genetic integrity during the vegetative growth of this species likely depends on mechanisms that act before hyphal fusion.     

Identifying the inserted locus of randomly integrated expression plasmids by whole-genome sequencing of Aspergillus strains

ABSTRACT

Whole-genome sequencing was conducted on two Aspergillus oryzae strains used for the manufacturing of food enzymes, Acrylaway® and Shearzyme®, with the aim of identifying the inserted locus of randomly integrated expression plasmid and obtaining flanking sequences for safety assessment. Illumina paired-end sequencing was employed, and the obtained reads were mapped to two references: the public genome sequence of Aspergillus oryzae RIB40 and the in-house sequence of the used expression plasmid. Introducing the concept of linking-reads, one locus for each was successfully identified as the integrated site. In the case of Acrylaway®, the obtained sequences suggested that the expression plasmid had been integrated as multiple copies in tandem form. In the case of Shearzyme®, however, information on one edge of the insert was missing, which required extra polymerase chain reaction (PCR) cloning for safety assessment. A 4-kb deletion was detected at the integrated site. There was also evidence of rearrangement occurring in Shearzyme® strain.     

Comparative Genome Analysis Between Aspergillus oryzae Strains Reveals Close Relationship Between Sites of Mutation Localization and Regions of Highly Divergent Genes among Aspergillus Species

Aspergillus oryzae has been utilized for over 1000 years in Japan for the production of various traditional foods, and a large number of A. oryzae strains have been isolated and/or selected for the effective fermentation of food ingredients. Characteristics of genetic alterations among the strains used are of particular interest in studies of A. oryzae. Here, we have sequenced the whole genome of an industrial fungal isolate, A. oryzae RIB326, by using a next-generation sequencing system and compared the data with those of A. oryzae RIB40, a wild-type strain sequenced in 2005. The aim of this study was to evaluate the mutation pressure on the non-syntenic blocks (NSBs) of the genome, which were previously identified through comparative genomic analysis of A. oryzae, Aspergillus fumigatus, and Aspergillus nidulans. We found that genes within the NSBs of RIB326 accumulate mutations more frequently than those within the SBs, regardless of their distance from the telomeres or of their expression level. Our findings suggest that the high mutation frequency of NSBs might contribute to maintaining the diversity of the A. oryzae genome.     

Improvement of Heterologous Protein Production in Aspergillus oryzae by RNA Interference with α-Amylase Genes

Aspergillus oryzae RIB40 has three α-amylase genes (amyA, amyB, and amyC), and secretes α-amylase abundantly. However, large amounts of endogenous secretory proteins such as α-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of α-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three α-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of α-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in α-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of α-amylase is effective in heterologous protein production in A. oryzae.     

菌株YN145对稻瘟病菌黑色素合成的影响及全基因组分析

【背景】枯草芽孢杆菌YN145是一株从湖南省桃江县的健康稻株中分离的细菌,前期研究中该菌对稻瘟病菌拮抗效果显著,在生物防治方面有很大的应用潜力。【目的】深入研究该菌株的生防机制并挖掘次级代谢产物基因资源。【方法】在4株稻瘟病菌生防菌中,选择其胞外抗菌物质抑制稻瘟病菌黑色素合成效果最佳的菌株YN145,采用紫外-可见分光光度计在波长400 nm处测定胞外和菌丝体内黑色素液的吸光度值,采用菌丝生长抑制平板法和分生孢子萌发抑制法测定抑菌活性。采用PacBio第三代测序和IlluminaHiSeq第二代测序相结合的技术对菌株YN145进行全基因组测序,并对测序数据进行组装,注释预测基因的功能,分析次级代谢产物合成基因簇。【结果】菌株YN145的胞外抗菌物质能较好地抑制稻瘟病菌黑色素合成、分生孢子萌发和菌丝生长。菌株YN145全基因组大小为4 167 871 bp,GC含量为43.86%,编码序列(coding sequence, CDS)数量为4 294个;共找到85个tRNA、30个rRNA和92个sRNA。同时预测到5个已知的次级代谢产物合成基因簇,分别编码合成bacillaene、bac...     

Perception of the chitin oligosaccharides contributes to disease resistance to blast fungus Magnaporthe oryzae in rice

Chitin is a component of fungal cell walls, and its fragments act as elicitors in many plants. The plasma membrane glycoprotein CEBiP, which possesses LysM domains, is a receptor for the chitin elicitor (CE) in rice. Here, we report that the perception of CE by CEBiP contributes to disease resistance against the rice blast fungus, Magnaporthe oryzae, and that enhanced responses to CE by engineering CEBiP increase disease tolerance. Knockdown of CEBiP expression allowed increased spread of the infection hyphae. To enhance defense responses to CE, we constructed chimeric genes composed of CEBiP and Xa21, which mediate resistance to rice bacterial leaf blight. The expression of either CRXa1 or CRXa3, each of which contains the whole extracellular portion of CEBiP, the whole intracellular domain of XA21, and the transmembrane domain from either CEBiP or XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after treatment with CE. Rice plants expressing the chimeric receptor exhibited necrotic lesions in response to CE and became more resistant to M. oryzae. Deletion of the first LysM domain in CRXA1 abolished these cellular responses. These results suggest that CEs are produced and recognized through the LysM domain of CEBiP during the interaction between rice and M. oryzae and imply that engineering pattern recognition receptors represents a new strategy for crop protection against fungal diseases.     

Endophytic fungus Falciphora oryzae promotes lateral root growth by producing indole derivatives after sensing plant signals

The endophytic fungus Falciphora oryzae was initially isolated from wild rice (Oryza granulata) and colonizes many crop species and promotes plant growth. However, the molecular mechanisms underlying F. oryzae-mediated growth promotion are still unknown. We found that F. oryzae was able to colonize Arabidopsis thaliana. The most dramatic change after F. oryzae inoculation was observed in the root architecture, as evidenced by increased lateral root growth but reduced primary root length, similar to the effect of auxin, a significant plant growth hormone. The expression of genes responsible for auxin biosynthesis, transport, and signalling was regulated in Arabidopsis roots after F. oryzae cocultivation. Indole derivatives were detected at significantly higher levels in liquid media after cocultivation compared with separate cultivation of Arabidopsis and F. oryzae. Consistently, the expression of indole biosynthetic genes was highly upregulated in F. oryzae upon treatment with Arabidopsis exudates. Global analysis of Arabidopsis gene expression at the early stage after F. oryzae cocultivation suggested that signals were exchanged to initiate Arabidopsis–F. oryzae interactions. All these results suggest that signalling molecules from Arabidopsis roots are perceived by F. oryzae and induce the biosynthesis of indole derivatives in F. oryzae, consequently stimulating Arabidopsis lateral root growth.     

利用二代测序技术高通量分析水稻品种间抗病性差异的分子基础

稻瘟病和白叶枯病是由稻温病菌(Magnaporthe oryzae)和白叶枯病菌(Xanthomonas oryzae pv.oryzae)引起的两种主要水稻病害,也是制约中国水稻生产的主要病害。为了从DNA水平探索造成水稻感病品种‘丽江新团黑谷’(LTH)和高抗品种‘特特普’(Tetep,TTP)间抗病性差异的分子基础,该研究对其已知的3个抗稻瘟病基因和3个抗白叶枯基因所在DNA区段分别进行PCR扩增,将等量混合的PCR产物再与基因组重测序样品按Ct值差值(ΔCt)~10的比例混合,采用二代测序技术进行一次性测序和比较分析,并对有差异的基因区域进行常规传统测序验证,以确定这2个品种中抗性基因(R基因)的数目和结构与品种抗病或感病表型的关联性。实验结果表明,二代测序能够快速并准确地寻找到2个不同水稻品种中多个特定基因的序列差异,且差异位点与常规测序结果相符。从LTH和TTP这2种抗性不同水稻品种在多个抗性基因的DNA水平差异来看,有差异的抗性基因位点在高抗品种TTP中大都与原始抗性基因序列相同,而对应的普感品种LTH的抗性基因往往多表现为氨基酸突变,这些序列差异很可能就是导致TTP与LTH抗性差异的分子基础。     

Identification of Sternbin and Naringenin as Detoxified Metabolites from the Rice Flavanone Phytoalexin Sakuranetin by Pyricularia oryzae

Sakuranetin ( 1 ) is a flavanone phytoalexin that has been reported to play an important role in disease resistance in rice plants. The rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae) has been reported to metabolize 1 to lower its antifungal activity. Here, two flavanones, sternbin ( 2 ) and naringenin ( 3 ), were identified as metabolites of 1 in P. oryzae suspension culture by liquid chromatography tandem mass spectrometry (LC/MS/MS). The inhibition of 1 , 2 , and 3 on P. oryzae mycelial growth were 45%, 19%, and 19%, respectively, at a concentration of 100 μm . Thus, 2 and 3 are detoxified metabolites of 1 by P. oryzae.     

Identification and characterization of suppressors of plant cell death (SPD) effectors from Magnaporthe oryzae

Phytopathogenic microorganisms, including the fungal pathogen Magnaporthe oryzae, secrete a myriad of effector proteins to facilitate infection. Utilizing the transient expression of candidate effectors in the leaves of the model plant Nicotiana benthamiana, we identified 11 suppressors of plant cell death (SPD) effectors from M. oryzae that were able to block the host cell death reaction induced by Nep1. Ten of these 11 were also able to suppress BAX‐mediated plant cell death. Five of the 11 SPD genes have been identified previously as either essential for the pathogenicity of M. oryzae, secreted into the plant during disease development, or as suppressors or homologues of other characterized suppressors. In addition, of the remaining six, we showed that SPD8 (previously identified as BAS162) was localized to the rice cytoplasm in invaded and surrounding uninvaded cells during biotrophic invasion. Sequence analysis of the 11 SPD genes across 43 re‐sequenced M. oryzae genomes revealed that SPD2, SPD4 and SPD7 have nucleotide polymorphisms amongst the isolates. SPD4 exhibited the highest level of nucleotide diversity of any currently known effector from M. oryzae in addition to the presence/absence polymorphisms, suggesting that this gene is potentially undergoing selection to avoid recognition by the host. Taken together, we have identified a series of effectors, some of which were previously unknown or whose function was unknown, that probably act at different stages of the infection process and contribute to the virulence of M. oryzae.