橡胶树暹罗炭疽菌Zn2Cys6型转录因子CsGcc1的生物学功能

【背景】暹罗炭疽菌（Colletotrichum siamense）是一种重要的病原真菌,可以引起炭疽病,给全球橡胶产业带来巨大的经济损失。Zn₂Cys₆型转录因子是真菌特有的锌指类转录因子,通常参与调控真菌的生长发育过程。【目的】在暹罗炭疽菌中鉴定了一个与稻瘟病菌Gcc1同源的Zn₂Cys₆型转录因子CsGcc1,并研究其功能。【方法】根据同源重组原理构建CsGCC1的基因敲除突变体,并通过营养生长、H₂O₂敏感性、分生孢子产生及萌发、玻璃纸试验和致病性分析,明确CsGcc1的功能。【结果】CsGcc1编码一个含有646个氨基酸的蛋白,而且含有一个GAL4结构域。CsGCC1基因在培养36 h的菌丝及分生孢子中具有较高的表达量。CsGCC1基因敲除突变株营养生长速率降低且对H₂O₂更加敏感。相较于野生型菌株,突变株的分生孢子产量、萌发率及附着胞形成率均降低。此外,CsGCC1的敲除可以明显降低分生孢子的穿透能力,突变株对橡胶叶片的致病力减弱。【结论】Zn₂Cys₆型转录因子CsGcc1参与调控暹罗炭疽菌的营养生长、氧化应激、分生孢子发育及致病性等过程。     

The Xanthomonas oryzae pv. oryzae PhoPQ two-component system is required for AvrXA21 activity, hrpG expression, and virulence

The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas oryzae pv. oryzae strains producing the type one system-secreted molecule, AvrXA21. X. oryzae pv. oryzae requires a regulatory two-component system (TCS) called RaxRH to regulate expression of eight rax (required for AvrXA21 activity) genes and to sense population cell density. To identify other key components in this critical regulatory circuit, we assayed proteins expressed in a raxR gene knockout strain. This survey led to the identification of the phoP gene encoding a response regulator that is up-regulated in the raxR knockout strain. Next we generated a phoP knockout strain and found it to be impaired in X. oryzae pv. oryzae virulence and no longer able to activate the response regulator HrpG (hypersensitive reaction and pathogenicity G) in response to low levels of Ca²⁺. The impaired virulence of the phoP knockout strain can be partially complemented by constitutive expression of hrpG, indicating that PhoP controls a key aspect of X. oryzae pv. oryzae virulence through regulation of hrpG. A gene encoding the cognate putative histidine protein kinase, phoQ, was also isolated. Growth curve analysis revealed that AvrXA21 activity is impaired in a phoQ knockout strain as reflected by enhanced growth of this strain in rice lines carrying XA21. These results suggest that the X. oryzae pv. oryzae PhoPQ TCS functions in virulence and in the production of AvrXA21 in partnership with RaxRH.     

A matter of structure: structural comparison of fungal carbonic anhydrases

Carbonic anhydrases (CAs) are metalloenzymes that catalyze the interconversion of carbon dioxide (CO₂) and hydrogen carbonate. CAs are distributed over all the three domains of life and are divided into five distinct evolutionarily unrelated gene families (α, β, γ, δ, ζ). In the large fungal kingdom, the majority of fungi encode multiple copies of β-CAs, with some also possessing genes for α-class CAs. Hemiascomycetous and basidiomycetous yeasts encode one or two β-CAs, while most of the filamentous ascomycetes have multiple copies of genes encoding α- and β-CAs. The functions of fungal β-CAs have been investigated intensively, while the role of fungal α-CAs is mostly unknown. The β-CAs are involved in sexual development, CO₂-sensing, pathogenicity, and survival in ambient air. Only recently, researchers have begun to use functional and structural data of CAs from pathogenic and non-pathogenic organisms to develop powerful and effective drugs and inhibitors or to identify enzymes that can be utilized in industrial applications. Despite the large number of fungal CAs known, only five have been characterized structurally: the α-CA AoCA of Aspergillus oryzae, the full length β-CA Can2 from the pathogenic basidiomycete Cryptococcus neoformans, the N-terminally truncated Saccharomyces cerevisiae β-CA Nce103, and two β-CAs of Sordaria macrospora. This review focuses on the functional and structural properties of fungal CAs.     

Fungus-Specific Sirtuin HstD Coordinates Secondary Metabolism and Development through Control of LaeA

The sirtuins are members of the NAD⁺-dependent histone deacetylase family that contribute to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungus-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungus-specific sirtuin HstD/Aspergillus oryzae Hst4 (AoHst4), which is a homolog of Hst4 in A. oryzae yeast. The deletion of all histone deacetylases in A. oryzae demonstrated that the fungus-specific sirtuin HstD/AoHst4 is required for the coordination of fungal development and secondary metabolite production. We also show that the expression of the laeA gene, which is the most studied fungus-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in a ΔhstD strain. Genetic interaction analysis of hstD/Aohst4 and laeA clearly indicated that HstD/AoHst4 works upstream of LaeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungus specific but conserved in the vast family of filamentous fungi. Thus, we conclude that the fungus-specific sirtuin HstD/AoHst4 coordinates fungal development and secondary metabolism via the regulation of LaeA in filamentous fungi.     

Bidirectional-Genetics Platform,a Dual-Purpose Mutagenesis Strategy for Filamentous Fungi

Rapidly increasing fungal genome sequences call for efficient ways of generating mutants to translate quickly gene sequences into their functions. A reverse genetic strategy via targeted gene replacement (TGR) has been inefficient for many filamentous fungi due to dominant production of undesirable ectopic transformants. Although large-scale random insertional mutagenesis via transformation (i.e., forward genetics) facilitates high-throughput uncovering of novel genes of interest, generating a huge number of transformants, which is necessary to ensure the likelihood of mutagenizing most genes, is time-consuming. We propose a new strategy, entitled the Bidirectional-Genetics (BiG) platform, which combines both forward and reverse genetic strategies by recycling ectopic transformants derived from TGR as a source for random insertional mutants. The BiG platform was evaluated using the rice blast fungus Magnaporthe oryzae as a model. Over 10% of >1,000 M. oryzae ectopic transformants, generated during disruption of specific genes, displayed abnormality in vegetative growth, pigmentation, and/or asexual reproduction. In this pool of putative mutants, we isolated insertional mutants with mutations in three genes involved in histidine biosynthesis (MoHIS5), vegetative growth (MoVPS74), or conidiophore formation (MoFRQ) (where “Mo” indicates “M. oryzae”), supporting the utility of this platform for systematic gene function studies.     

Pex14/17, a filamentous fungus‐specific peroxin,is required for the import of peroxisomal matrix proteins and full virulence of Magnaporthe oryzae

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfil a variety of biochemical functions. The biogenesis of peroxisomes requires a variety of proteins, named peroxins, which are encoded by PEX genes. Pex14/17 is a putative recently identified peroxin, specifically present in filamentous fungal species. Its function in peroxisomal biogenesis is still obscure and its roles in fungal pathogenicity have not yet been documented. Here, we demonstrate the contributions of Pex14/17 in the rice blast fungus Magnaporthe oryzae (Mopex14/17) to peroxisomal biogenesis and fungal pathogenicity by targeting gene replacement strategies. Mopex14/17 has properties of both Pex14 and Pex17 with regard to its protein sequence. Mopex14/17 is distributed at the peroxisomal membrane and is essential for efficient peroxisomal targeting of proteins containing peroxisomal targeting signal 1. MoPEX19 deletion leads to the cytoplasmic distribution of Mopex14/17, indicating that the peroxisomal import of Pex14/17 is dependent on Pex19. The knockout mutants of MoPEX14/17 show reduced fatty acid utilization, reactive oxygen species (ROS) degradation and cell wall integrity. Moreover, Δmopex14/17 mutants show delayed conidial generation and appressorial formation, and a reduction in appressorial turgor accumulation and penetration ability in host plants. These defects result in a significant reduction in the virulence of the mutant. These data indicate that MoPEX14/17 plays a crucial role in peroxisome biogenesis and contributes to fungal development and pathogenicity.     

Alanine: Glyoxylate aminotransferase 1 is required for mobilization and utilization of triglycerides during infection process of the rice blast pathogen,Magnaporthe oryzae

The rice blast pathogen, Magnaporthe oryzae has been widely used as a model pathogen to study plant infection-related fungal morphogenesis, such as penetration via appressorium and plant-microbe interactions at the molecular level. Previously, we identified a gene encoding peroxisomal alanine: glyoxylate aminotransferase 1 (AGT1) in M. oryzae and demonstrated that the AGT1 was indispensable for pathogenicity. The AGT1 knockout mutants were unable to penetrate the host plants, such as rice and barley, and therefore were non-pathogenic. The inability of ∆Moagt1 mutants to penetrate the susceptible plants was likely due to the disruption in coordination of the β-oxidation and the glyoxylate cycle resulted from a blockage in lipid droplet mobilization and eventually utilization during conidial germination and appressorium morphogenesis, respectively. Here, we further demonstrate the role of AGT1 in lipid mobilization by in vitro germination assays and confocal microscopy.     

Autophagy vitalizes the pathogenicity of pathogenic fungi

Plant pathogenic fungi utilize a series of complex infection structures, in particular the appressorium, to gain entry to and colonize plant tissue. As a consequence of the accumulation of huge quantities of glycerol in the cell the appressorium generates immense intracellular turgor pressure allowing the penetration peg of the appressorium to penetrate the leaf cuticle. Autophagic processes are ubiquitous in eukaryotic cells and facilitate the bulk degradation of macromolecules and organelles. The study of autophagic processes has been extended from the model yeast Saccharomyces cerevisiae to pathogenic fungi such as the rice blast fungus Magnaporthe oryzae. Significantly, null mutants for the expression of M. oryzae autophagy gene homologs lose their pathogenicity for infection of host plants. Clarification of the functions and network of interactions between the proteins expressed by M. oryzae autophagy genes will lead to a better understanding of the role of autophagy in fungal pathogenesis and help in the development of new strategies for disease control.