Effectors of biotrophic fungi and oomycetes: pathogenicity factors and triggers of host resistance

Many biotrophic fungal and oomycete pathogens share a common infection process involving the formation of haustoria, which penetrate host cell walls and form a close association with plant membranes. Recent studies have identified a class of pathogenicity effector proteins from these pathogens that is transferred into host cells from haustoria during infection. This insight stemmed from the identification of avirulence (Avr) proteins from these pathogens that are recognized by intracellular host resistance (R) proteins. Oomycete effectors contain a conserved translocation motif that directs their uptake into host cells independently of the pathogen, and is shared with the human malaria pathogen. Genome sequence information indicates that oomycetes may express several hundred such host-translocated effectors. Elucidating the transport mechanism of fungal and oomycete effectors and their roles in disease offers new opportunities to understand how these pathogens are able to manipulate host cells to establish a parasitic relationship and to develop new disease-control measures.     

Protein complexes mediate signalling in plant responses to hormones,light, sucrose and pathogens

Living organisms use complex pathways of signal perception and transduction to respond to stimuli in their environments. In plants, putative signal transduction components have been identified through mutant screens and comparative analysis of genome sequences of model eukaryotes. Several pieces in a large series of puzzles have now been identified and a current challenge is to determine how these pieces interconnect. Functional analysis of the encoded proteins has necessitated a change from genetic to biochemical approaches. In recent years, the application of techniques such as two-hybrid screening and epitope tagging has facilitated the study of protein-protein interactions and has increased our understanding of cellular signalling mechanisms. One focus of present research is the ubiquitin/proteasome-mediated degradation of proteins. Increasing evidence suggests this is a control common to many plant signalling pathways including development and responsiveness to hormones, light and sucrose. A central challenge in the study of plant disease resistance has been to identify protein complexes that contain host defence proteins and pathogenicity factors. In this review we summarize the latest developments in these areas where the existence of protein complexes has been demonstrated to be of fundamental importance in plant signalling.     

山田胶锈菌和亚洲胶锈菌吸器的比较转录组分析

为防止锈病的传播,培育抗病品种以及减少产量损失,基于山田胶锈菌(Gymnosporangium yamadae)和亚洲胶锈菌(Gymnosporangium asiaticum)吸器阶段的转录组差异分析揭示了胶锈菌侵染寄主植物时的专化性选择机制。对山田胶锈菌和亚洲胶锈菌担孢子侵染寄主时形成的吸器进行转录组测序,分别获得了21 213条和13 015条单基因(unigenes);从山田胶锈菌和亚洲胶锈菌中分别选择5个基因进行实时荧光定量PCR验证,显示其表达情况与转录组分析结果基本一致,表明转录组分析结果可靠;用Nr、GO、KEGG、KOG等7个数据库进行基因功能注释和富集分析,发现两种胶锈菌的基因主要富集在细胞进程、翻译、代谢相关通路;使用SignalP和TMHMM在线网站以及dbCAN、BLAST、HMMER等软件分析显示山田胶锈菌和亚洲胶锈菌吸器中的候选效应蛋白分别有343个(2.51%)和175个(2.79%),其中分别含有14个和5个蛋白酶,10个和3个脂酶;利用OrthoFinder、BLSAT和KaKs Calculator软件分析了两种胶锈菌的进化关系,在一对一同源基因中...     

植物抗病基因(R)与病原物无毒基因(Avr)相互作用机制的研究进展

PTI和ETI是植物在长期进化过程中形成的两类抵抗病原物的机制。基因对基因假说的抗病方式属于ETI抗性机制的一种,该假说认为具有保守NB-LRR结构域的R蛋白识别病原物非保守的无毒蛋白效应子(Avr),激活防卫反应信号途径,导致过敏性坏死。植物抗病基因(R)与病原菌无毒基因(Avr)产物间的直接或间接相互作用而产生的基因对基因抗性是植物抗病性的重要形式,该文对植物抗病蛋白与无毒蛋白相互作用机制进行了综述。其中,间接相互作用模式是主要方式。     

Fungal Avirulence Genes: Structure and Possible Functions

Avirulence (Avr) genes exist in many fungi that share a gene-for-gene relationship with their host plant. They represent unique genetic determinants that prevent fungi from causing disease on plants that possess matching resistance (R) genes. Interaction between elicitors (primary or secondary products ofAvrgenes) and host receptors in resistant plants causes induction of various defense responses often involving a hypersensitive response.Avrgenes have been successfully isolated by reverse genetics and positional cloning. Five cultivar-specificAvrgenes (Avr4,Avr9, andEcp2 fromCladosporium fulvum; nip1fromRhynchosporium secalis;andAvr2-YAMOfromMagnaporthe grisea) and three species-specificAvrgenes (PWL1andPWL2fromM. griseaandinf1fromPhytophthora infestans) have been cloned. Isolation of additionalAvrgenes from these fungi, but also from other fungi such asUromyces vignae,Melampsora lini, Phytophthora sojae,andLeptosphaeria maculans,is in progress. Molecular analyses of nonfunctionalAvrgene alleles show that these originate from deletions or mutations in the open reading frame or the promoter sequence of anAvrgene. Although intrinsic biological functions of mostAvrgene products are still unknown, recent studies have shown that twoAvrgenes,nip1andEcp2, encode products that are important pathogenicity factors. All fungalAvrgenes cloned so far have been demonstrated or predicted to encode extracellular proteins. Current studies focus on unraveling the mechanisms of perception of avirulence factors by plant receptors. The exploitation ofAvrgenes and the matchingRgenes in engineered resistance is also discussed.     

PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [¹⁴C]‐acetyl‐CoA to oligogalacturonides. Through site‐directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting the acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5 and pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibile to multiple strains of the generalist necrotroph Botrytis cinerea, and have decreased camalexin production upon infection with B. cinerea. These results illustrate that cell wall composition is intimately connected to fungal disease resistance and outline a potential route for engineering powdery mildew resistance into susceptible crop species.     

植物抗病基因与病原菌无毒基因互作的分子基础

近10年来,大量的植物抗病基因和病原菌无毒基因被克隆,抗病基因和无毒基因的结构、功能及其互作关系的研究也取得重大进展。通过介绍抗病基因与无毒基因互作的两种模式,从抗病基因与病原菌无毒基因互作角度探讨了抗病基因在植物抗病育种和农作物生产中直用的问题,提出抗细菌和真菌单基因转化很难赋予农作物切实抗性。     

植物抗病蛋白研究进展

为了应对外界复杂的环境变化, 植物进化出一套复杂而精细的免疫应答调控机制。植物抗病蛋白能够特异地识别病原微生物分泌的效应蛋白, 触发免疫响应以对抗病原微生物的侵扰。该文综述了植物抗病蛋白的结构与功能及对病原菌的识别方式、在免疫响应过程中抗病蛋白的动态平衡机制及其介导的防御反应信号转导。开展植物抗病蛋白研究可为定向培育抗病作物奠定理论基础。     

Effect of Host Plant Resistance on Haustorium Formation in Cereal Rust Fungi

Haustoria of Puccinia triticina (wheat leaf rust fungus) and P. hordei (barley leaf rust fungus) were isolated from susceptible and partially resistant wheat lines, and susceptible, hypersensitive and partially resistant barley lines. Haustoria were counted and measured. The size of haustoria was similar in the partially resistant and susceptible genotypes but haustoria were smaller in the hypersensitive barley line L94+Pa7. The number of haustoria was reduced in both partially and hypersensitive lines when compared with susceptible ones. Therefore it seems that the reduction in the number of haustoria is a consequence of the resistance that can be attributable either to early abortion of infection units or reduced colony growth. The reduction of the number of haustoria was more pronounced in the adult plant stage.     

Beetle grazing reduces natural infection of Rumex obtusifolius by fungal pathogens

In previously reported laboratory experiments, infection of Rumex obtusifolius by the rust fungus Uromyces rumicis was decreased on leaves which had prior herbivory by the beetle Gastrophysa viridula . In this paper we investigate whether this interaction is robust for natural infection by a variety of fungi in field experiments carried out in spring and autumn with plants given different levels of nitrogen fertilization. Grazing by G. viridula led to a decrease in lesion density of Ramularia rubella and Venturia rumicis in the spring and V. rumicis and U. rumicis in the autumn experiment. For V. rumicis and U. rumicis significant reductions in lesion density occurred on the undamaged leaves of damaged plants, compared with similar leaves on undamaged plants, suggesting systemic induced resistance. This induced resistance was usually independent of the amount of nitrogen fertilization, although the inhibitory effect of grazing on R. rubella in the spring and V. rumicis in the autumn experiment was enhanced by increasing nitrogen fertilization and was inhibited by increasing nitrogen fertilization for V. rumicis in the spring. In both experiments, the lesion density of V. rumicis was greater on leaves on which R. rubella was also present, and the presence of U. rumicis in the autumn experiment was linked to a similar but greater effect on V. rumicis lesion density. We found no evidence of induced resistance by fungi against fungi in these experiments. We highlight the complex interactions between inhibitory and facilitatory processes acting on leaf fungal infection. These results are compared with the proposed molecular mechanisms of induced resistance(s) and we consider the benefits of closer integration between molecular and ecological investigations of induced resistances that occur in the field.     

Involvement of lipid transfer proteins in resistance against a non-host powdery mildew in Arabidopsis thaliana

Non-specific lipid transfer proteins (LTPs) are involved in the transport of lipophilic compounds to the cuticular surface in epidermal cells and in the defence against pathogens. The role of glycophosphatidylinositol (GPI)-anchored LTPs (LTPGs) in resistance against non-host mildews in Arabidopsis thaliana was investigated using reverse genetics. Loss of either LTPG1, LTPG2, LTPG5 or LTPG6 increased the susceptibility to penetration of the epidermal cell wall by Blumeria graminis f. sp. hordei (Bgh). However, no impact on pre-penetration defence against another non-host mildew, Erysiphe pisi (Ep), was observed. LTPG1 was localized to papillae at the sites of Bgh penetration. This study shows that, in addition to the previously known functions, LTPGs contribute to pre-invasive defence against certain non-host powdery mildew pathogens.     

一粒小麦抗白粉病和条锈病基因的分析

一粒小麦是普通小麦抗性改良的宝贵资源.本研究对24份一粒小麦分别进行了白粉病和条锈病混合菌种苗期接种鉴定,进一步分别用一套白粉病菌菌株(15个)对2份乌拉尔图小麦和条锈病菌小种(21个)对1份栽培一粒小麦进行接种鉴定,其中乌拉尔图小麦UR206能抵抗所有供试白粉菌菌株,UR204除对白粉菌菌株E11感病外,对其余菌株表现抗性;栽培一粒小麦MO205对不同条锈菌小种表现出不同的抗性反应,研究表明乌拉尔图小麦UR206、UR204和栽培一粒小麦MO205分别含有与已知抗白粉病和抗条锈病基因不同的新基因.对乌拉尔图小麦UR204、UR206和栽培一粒小麦MO205分别进行抗白粉和条锈病基因的遗传分析,结果表明乌拉尔图小麦UR204和UR206分别含有一对显性抗白粉病基因,栽培一粒小麦MO205含有两对独立遗传的显性抗条锈病基因.     

High disease incidence and apparent disease tolerance in a North American Great Basin plant community

Patterns and consequences of plant disease at the community level have rarely been studied. We surveyed fungal infection in a Great Basin community of perennial shrubs over 4 years. Repeat surveys in fixed plots and along transects showed that disease incidence in the dominant perennial species was often very high, with up to 100% of all individuals infected. Despite the widespread prevalence of infection, and its severity on individual plants (which sometimes had over 1/3 of their leaves covered in pustules), its effects on survival and flowering were undetectably small. Thus, this perennial community appears to be stable, despite widespread disease. There are two potential explanations for this pattern; either the pathogens have evolved to be avirulent, or the hosts have become tolerant to being infected. Avirulence is not likely, because multiple infections are common in this system, and multiple infections have been shown in other species to favor strains that are faster reproducing and thus more virulent. Instead, it is more likely that tolerance has evolved in these host species, because infection in each year is practically inevitable and because the host plants are long-lived, giving little opportunity for new resistance genotypes to evolve. This revised version was published online in July 2006 with corrections to the Cover Date.     

Proteinaceous effector discovery and characterization in filamentous plant pathogens

The complicated interplay of plant–pathogen interactions occurs on multiple levels as pathogens evolve to constantly evade the immune responses of their hosts. Many economically important crops fall victim to filamentous pathogens that produce small proteins called effectors to manipulate the host and aid infection/colonization. Understanding the effector repertoires of pathogens is facilitating an increased understanding of the molecular mechanisms underlying virulence as well as guiding the development of disease control strategies. The purpose of this review is to give a chronological perspective on the evolution of the methodologies used in effector discovery from physical isolation and in silico predictions, to functional characterization of the effectors of filamentous plant pathogens and identification of their host targets.     

Extracellular transport and integration of plant secretory proteins into pathogen-induced cell wall compartments

Many fungal parasites enter plant cells by penetrating the host cell wall and, thereafter, differentiate specialized intracellular feeding structures, called haustoria, by invagination of the plant's plasma membrane. Arabidopsis PEN gene products are known to act at the cell periphery and function in the execution of apoplastic immune responses to limit fungal entry. This response underneath fungal contact sites is tightly linked with the deposition of plant cell wall polymers, including PMR4/GSL5-dependent callose, in the paramural space, thereby producing localized wall thickenings called papillae. We show that powdery mildew fungi specifically induce the extracellular transport and entrapment of the fusion protein GFP–PEN1 syntaxin and its interacting partner monomeric yellow fluorescent protein (mYFP)–SNAP33 within the papillary matrix. Remarkably, PMR4/GSL5 callose, GFP–PEN1, mYFP–SNAP33, and the ABC transporter GFP–PEN3 are selectively incorporated into extracellular encasements surrounding haustoria of the powdery mildew Golovinomyces orontii , suggesting that the same secretory defense responses become activated during the formation of papillae and haustorial encasements. This is consistent with a time-course analysis of the encasement process, indicating that these extracellular structures are generated through the extension of papillae. We show that PMR4/GSL5 callose accumulation in papillae and haustorial encasements occurs independently of PEN1 syntaxin. We propose a model in which exosome biogenesis/release serves as a common transport mechanism by which the proteins PEN1 and PEN3, otherwise resident in the plasma membrane, together with membrane lipids, become stably incorporated into both pathogen-induced cell wall compartments.     

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol‐3‐phosphate‐binding proteins

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol‐3‐phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P‐binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P‐binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P‐binding site, or by a secreted PI4P‐binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P‐binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P‐binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.     

Puccinia coronata f. sp. avenae: a threat to global oat production

Puccinia coronata f. sp. avenae (Pca) causes crown rust disease in cultivated and wild oat (Avena spp.). The significant yield losses inflicted by this pathogen make crown rust the most devastating disease in the oat industry. Pca is a basidiomycete fungus with an obligate biotrophic lifestyle, and is classified as a typical macrocyclic and heteroecious fungus. The asexual phase in the life cycle of Pca occurs in oat, whereas the sexual phase takes place primarily in Rhamnus species as the alternative host. Epidemics of crown rust happens in areas with warm temperatures (20–25 °C) and high humidity. Infection by the pathogen leads to plant lodging and shrivelled grain of poor quality. Disease symptoms : Infection of susceptible oat varieties gives rise to orange–yellow round to oblong uredinia (pustules) containing newly formed urediniospores. Pustules vary in size and can be larger than 5 mm in length. Infection occurs primarily on the surfaces of leaves, although occasional symptoms develop in the oat leaf sheaths and/or floral structures, such as awns. Symptoms in resistant oat varieties vary from flecks to small pustules, typically accompanied by chlorotic halos and/or necrosis. The pycnial and aecial stages are mostly present in the leaves of Rhamnus species, but occasionally symptoms can also be observed in petioles, young stems and floral structures. Aecial structures display a characteristic hypertrophy and can differ in size, occasionally reaching more than 5 mm in diameter. Taxonomy : Pca belongs to the kingdom Fungi, phylum Basidiomycota, class Pucciniomycetes, order Pucciniales and family Pucciniaceae. Host range : Puccinia coronata sensu lato can infect 290 species of grass hosts. Pca is prevalent in all oat‐growing regions and, compared with other cereal rusts, displays a broad telial host range. The most common grass hosts of Pca include cultivated hexaploid oat (Avena sativa) and wild relatives, such as bluejoint grass, perennial ryegrass and fescue. Alternative hosts include several species of Rhamnus, with R. cathartica (common buckthorn) as the most important alternative host in Europe and North America. Control : Most crown rust management strategies involve the use of rust‐resistant crop varieties and the application of fungicides. The attainment of the durability of resistance against Pca is difficult as it is a highly variable pathogen with a great propensity to overcome the genetic resistance of varieties. Thus, adult plant resistance is often exploited in oat breeding programmes to develop new crown rust‐resistant varieties. Useful website : https://www.ars.usda.gov/midwest-area/st-paul-mn/cereal-disease-lab/docs/cereal-rusts/race-surveys/ .     

Host and non-host pathogens elicit different jasmonate/ethylene responses in Arabidopsis

Arabidopsis does not support the growth and asexual reproduction of the barley pathogen, Blumeria graminis f. sp. hordei Bgh). A majority of germlings fail to penetrate the epidermal cell wall and papillae. To gain additional insight into this interaction, we determined whether the salicylic acid (SA) or jasmonate (JA)/ethylene (ET) defence pathways played a role in blocking barley powdery mildew infections. Only the eds1 mutant and NahG transgenics supported a modest increase in penetration success by the barley powdery mildew. We also compared the global gene expression patterns of Arabidopsis inoculated with the non-host barley powdery mildew to those inoculated with a virulent, host powdery mildew, Erysiphe cichoracearum. Genes repressed by inoculations with non-host and host powdery mildews relative to non-inoculated control plants accounted for two-thirds of the differentially expressed genes. A majority of these genes encoded components of photosynthesis and general metabolism. Consistent with this observation, Arabidopsis growth was inhibited following inoculation with Bgh, suggesting a shift in resource allocation from growth to defence. A number of defence-associated genes were induced during both interactions. These genes likely are components of basal defence responses, which do not effectively block host powdery mildew infections. In addition, genes encoding defensins, anti-microbial peptides whose expression is under the control of the JA/ET signalling pathway, were induced exclusively by non-host pathogens. Ectopic activation of JA/ET signalling protected Arabidopsis against two biotrophic host pathogens. Taken together, these data suggest that biotrophic host pathogens must either suppress or fail to elicit the JA/ET signal transduction pathway.     

甜瓜抗霜霉病基因同源序列克隆与分析

采用RT—PCR扩增的方法,从高抗霜霉病甜瓜品种‘日本安农二号’中克隆到约3kb的cDNA片段（命名为MRGH-D,该基因是一个连续的通读编码框,编码1007个氨基酸。推测的蛋白质分子量为113．7kDa,等电点为7．88,蛋白质预测无跨膜区。根据推测的氨基酸序列,该基因属于TIR—NBS—LRR类抗病基因,具有TIR-NBS—LRR类抗病基因所有的保守结构域。核苷酸序列和氨基酸序列同源性分析结果显示,MRGH-J与甜瓜抗病基因的同源序列MRGHl2及抗霜霉病相关基因mp-19均具有高达99％的同源性,推测该基因可能在甜瓜抗霜霉病中起作用。