The hemibiotrophic lifestyle of Colletotrichum species

Colletotrichum species infect several economically important crop plants. To establish a compatible parasitic interaction, a specialized infection cell, the melanized appressorium, is differentiated on the cuticle of the host. After penetration, an infection vesicle and primary hyphae are formed. These structures do not kill the host cell and show some similarities with haustoria formed by powdery mildews and rust fungi. Therefore, this stage of infection is called biotrophic. Later in the infection process, necrotrophic secondary hyphae spread within and kill the host tissue. The lifestyle of Colletotrichum species is called hemibiotrophic, as biotrophic and necrotrophic developmental stages are sequentially established. As most Colletotrichum species are accessible to molecular techniques, genes can be identified and functionally characterized. Here we demonstrate that Agrobacterium tumefaciens-mediated transformation is a well-suited method for tagging of genes mediating compatibility in the Colletotrichum graminicola–maize interaction.     

Plant infection and the establishment of fungal biotrophy

To exploit plants as living substrates, biotrophic fungi have evolved remarkable variations of their tubular cells, the hyphae. They form infection structures such as appressoria, penetration hyphae and infection hyphae to invade the plant with minimal damage to host cells. To establish compatibility with the host, controlled secretory activity and distinct interface layers appear to be essential. Colletotrichum species switch from initial biotrophic to necrotrophic growth and are amenable to mutant analysis and molecular studies. Obligate biotrophic rust fungi can form the most specialized hypha: the haustorium. Gene expression and immunocytological studies with rust fungi support the idea that the haustorium is a transfer apparatus for the long-term absorption of host nutrients.     

Colletotrichum: A model genus for studies on pathology and fungal-plant interactions.

Species of Colletotrichum use diverse strategies for invading host tissue, ranging from intracellular hemibiotrophy to subcuticular intramural necrotrophy. In addition, these pathogens develop a series of specialized infection structures, including germ tubes, appressoria, intracellular hyphae, and secondary necrotrophic hyphae. Colletotrichum species provide excellent models for studying the molecular basis of infection structure differentiation and fungal-plant interactions. In this review we cover the various stages of the infection processes of Colletotrichum species, including spore adhesion and germination, germ tube and appressorium differentiation and functions, and biotrophic and necrotrophic development. The contribution of molecular, biochemical, and immunological approaches to the identification of genes and proteins relevant to each stage of fungal development will be considered. As well as reviewing results from several groups, we also describe our own work on the hemibiotrophic pathogen, C. lindemuthianum.     

Infection Structure–Specific Expression of β-1,3-Glucan Synthase Is Essential for Pathogenicity of Colletotrichum graminicola and Evasion of β-Glucan–Triggered Immunity in Maize

β-1,3-Glucan and chitin are the most prominent polysaccharides of the fungal cell wall. Covalently linked, these polymers form a scaffold that determines the form and properties of vegetative and pathogenic hyphae. While the role of chitin in plant infection is well understood, the role of β-1,3-glucan is unknown. We functionally characterized the β-1,3-glucan synthase gene GLS1 of the maize (Zea mays) pathogen Colletotrichum graminicola, employing RNA interference (RNAi), GLS1 overexpression, live-cell imaging, and aniline blue fluorochrome staining. This hemibiotroph sequentially differentiates a melanized appressorium on the cuticle and biotrophic and necrotrophic hyphae in its host. Massive β-1,3-glucan contents were detected in cell walls of appressoria and necrotrophic hyphae. Unexpectedly, GLS1 expression and β-1,3-glucan contents were drastically reduced during biotrophic development. In appressoria of RNAi strains, downregulation of β-1,3-glucan synthesis increased cell wall elasticity, and the appressoria exploded. While the shape of biotrophic hyphae was unaffected in RNAi strains, necrotrophic hyphae showed severe distortions. Constitutive expression of GLS1 led to exposure of β-1,3-glucan on biotrophic hyphae, massive induction of broad-spectrum defense responses, and significantly reduced disease symptom severity. Thus, while β-1,3-glucan synthesis is required for cell wall rigidity in appressoria and fast-growing necrotrophic hyphae, its rigorous downregulation during biotrophic development represents a strategy for evading β-glucan–triggered immunity.     

The biotrophy-associated secreted protein 4 (BAS4) participates in the transition of Magnaporthe oryzae from the biotrophic to the necrotrophic phase

The physiological and metabolic processes of host plants are manipulated and remodeled by phytopathogenic fungi during infection, revealed obvious signs of biotrophy of the hemibiotrophic pathogen. As we known that effector proteins play key roles in interaction of hemibiotrophic fungi and their host plants. BAS4 (biotrophy-associated secreted protein 4) is an EIHM (extrainvasive hyphal membrane) matrix protein that was highly expressed in infectious hyphae. In order to study whether BAS4 is involved in the transition of rice blast fungus from biotrophic to necrotrophic phase, The susceptible rice cultivar Lijiangxintuanheigu (LTH) that were pre-treated with prokaryotic expression product of BAS4 and then followed with inoculation of the blast strain, more serious blast disease symptom, more biomass such as sporulation and fungal relative growth, and lower expression level of pathogenicity-related genes appeared in lesion of the rice leaves than those of the PBS-pretreated-leaves followed with inoculation of the same blast strain, which demonstrating that BAS4 invitro changed rice defense system to facilitate infection of rice blast strain. And the susceptible rice cultivar (LTH) were inoculated withBAS4-overexpressed blast strain, we also found more serious blast disease symptom and more biomass also appeared in lesion of leaves inoculated with BAS4-overexpressed strain than those of leaves inoculated with the wild-type strain, and expression level of pathogenicity-related genes appeared lower in biotrophic phase and higher in necrotrophic phase of infection, indicating BAS4 maybe in vivo regulate defense system of rice to facilitate transition of biotrophic to necrotrophic phase. Our data demonstrates that BAS4 in vitro and in vivo participates in transition from the biotrophic to the necrotrophic phase of Magnaporthe oryzae.     

A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions

The ability of a Colletotrichum sp., originally isolated from Brassica campestris, to infect Arabidopsis thaliana was examined. Sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal (r)DNA showed the pathogen to be Colletotrichum destructivum. The host range was broad, including many cruciferous plants and some legumes. At 25 degrees C, all A. thaliana accessions tested were susceptible to the Brassica isolates of C. destructivum; however, at 15 degrees C, the accession Ws-2 showed a temperature-dependant resistance, in which single epidermal cells underwent a rapid hypersensitive response. Legume isolates of C. destructivum were unable to infect A. thaliana and induced deposition of callose papillae at sites of attempted penetration. In compatible interactions, C. destructivum showed a two-stage, hemibiotrophic infection process. The initial biotrophic phase was associated with large, intracellular primary hyphae and was confined to one epidermal cell; whereas, in the subsequent necrotrophic phase, narrow secondary hyphae extensively colonized the tissue and conidia were produced in acervuli. An efficient transformation system was established for C. destructivum, using Agrobacterium-mediated transfer of DNA. The ability to genetically manipulate both partners in the interaction is an important advantage, and the Arabidopsis-Colletotrichum pathosystem should provide a valuable new model for dissecting plant-fungal interactions.     

Increased expression of a plant actin gene during a biotrophic interaction between round-leaved mallow, Malva pusilla, and Colletotrichum gloeosporioides f. sp. malvae

Two actin genes, actA from the hemibiotrophic anthracnose fungus, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp. malvae, and act1 from its host, Malva pusilla (Sm.) were cloned from a cDNA library developed from infected host tissue. The actin gene, actA, of C. gloeosporioides f. sp. malvae, which is similar to that of other euascomycetes, appears to be expressed constitutively. The actin gene of M. pusilla is most similar to one of the actin genes of Arabidopsis thaliana that is unique in being responsive to environmental stimuli such as wounding. Expression of actA was used to follow the growth of the fungus in the plant tissue. Low actA expression occurred until 72–96 h after inoculation and then increased rapidly, corresponding with the timing of the shift from slower biotrophic fungal growth to much more rapid necrotrophic growth. In contrast, expression of act1 approximately doubled during the biotrophic phase and then rapidly declined during the necrotrophic phase. Increased host actin expression could be due to host cytoskeleton rearrangement in response to biotrophic infection, and the subsequent decrease in host actin expression could be due to host cell disruption resulting from tissue maceration during necrosis. This is the first report of a host actin gene that can increase in expression during a compatible plant-pathogen interaction. Received: 15 March 1999 / Accepted: 1 May 1999     

柿树炭疽菌侵染寄主的细胞学研究*

超微结构研究表明,柿树炭疽菌(Colletotrichum gloeosporioides)侵染后在寄主细胞中形成初生菌丝和次生菌丝,寄主细胞膜外沉积了一层厚的电子不透明物质,初生菌丝与具有沉积物的寄主原生质膜之间有一层界面基质(interfacial matrix)。当初生菌丝扩张并侵染相邻细胞时, 围绕着初生菌丝层的界面基质消失,具有沉积物的原生质膜被逐步降解。初生菌丝在穿透寄主细胞壁过程中形成一个漏斗状的菌丝锥,然后穿透寄主细胞壁并迅速膨大, 然后形成厚壁的初生菌丝。初生菌丝在寄主细胞壁中收缩狭窄处产生一个隔膜,隔膜两边菌丝中细胞质的电子密度明显不同,菌丝锥中有浓密的电子密度。死体营养的次生菌丝在死的细胞中繁殖和扩展,并产生分枝。次生菌丝可直接穿透较薄的寄主细胞壁,无缢缩或任何变形现象,菌丝顶端部分未见隔膜产生;在穿透较厚的细胞壁时,靠近顶端处产生隔膜,顶端细胞膨大,使寄主细胞壁撕裂。接种90h后分生孢子盘在枝条表面形成。柿树炭疽菌其侵染过程有两个阶段,即初生菌丝的活体营养阶段和次生菌丝的死体营养阶段。     

Mycoparasitism within the Zygomycetes

The Zygomycetes includes a number of mycoparasitic genera, which differ in their strategies of parasitism. Piptocephalis, Dispira, Dimargaris and Tieghemiomyces are typical biotrophs, and display many features associated with this mode of infection, such as the formation of haustoria. Dicranophora, Spinellus and Sylgiles, on the other hand, apparently form necrotrophic associations with moribund toadstools, although it is difficult to define the boundary between mycoparasitism and competitive saprophytism. There are also zygomycetes, such as Chaetocladium and Syncephalis, which have modes of infection which do not fit neatly into either category above, but apparently share necrotrophic and biotrophic characteristics. Initially the infection process of Syncephalis resembles that of Piptocephalis, but it is followed by a rapid internal growth of parasitic hyphae and concomitant destruction of host cytoplasm. Chaetocladium forms gall-like structures on suitable host fungi and its growth is enhanced by this association. Circumstantial evidence suggests that these galls are functionally different from those formed by Parasitella during a pseudo-sexual response to the presence of another fungus. Zygomycetes also act as hosts for several other mycoparasitic fungi.     

Infection induced defence responses in sorghum,with special emphasis on accumulation of reactive oxygen species and cell wall modifications

Hemibiotrophic plant pathogens pass both biotrophic and necrotrophic phases during their infection cycle. Colletotrichum spp. exhibits two types of hemibiotrophy, i.e. intracellular and subcuticular intramural. Colletotrichum sublineolum infecting sorghum exhibits a typical intracellular type of hemibiotrophy. During the biotrophic stage of C. sublineolum infection in sorghum, several cell wall-associated defence reactions are activated and efficiently participate in stopping pathogen development. Among these defence reactions, generation and accumulation of reactive oxygen species (ROS) and cell wall barrier formation are key factors in resistance. This review focuses on the infection processes of C. sublineolum in sorghum and the defence responses activated, with special emphasis on accumulation of ROS and hydroxy-rich glyco-proteins (HRGPs).     

The role of ethylene during the infection of Nicotiana tabacum by Colletotrichum destructivum

Two periods of increased ethylene production were observed after inoculation of Nicotiana tabacum by Colletotrichum destructivum. This pathogen exhibits an intracellular hemibiotrophic infection process, with a biotrophic phase followed by a necrotrophic phase. Ethylene production first increased during the biotrophic phase with a peak at 24 h before the necrotrophic phase. A second increase in ethylene occurred late in the necrotrophic phase when the lesions were expanding. Two different 1-aminocyclopropane-1-carboxylic acid synthase genes showed increased expression after the first ethylene peak with a maximum at 24 h before the second ethylene increase. Expression of an 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) gene increased during the first ethylene peak and then declined at the beginning of the second ethylene increase. A second ACO gene showed relatively little change in expression during infection with slightly higher expression at 24 h before the second ethylene increase, and a third ACO gene showed a progressive decline in expression with a major decrease occurring before the second ethylene increase. Inoculation of ethylene-insensitive tobacco with C. destructivum revealed that it was more susceptible than the wild type. The changes in ethylene production and associated gene expression as well as the increased disease susceptibility of ethylene-insensitive tobacco indicate that ethylene plays a role in this interaction, perhaps as a signalling molecule to trigger defense mechanisms.