Class V chitin synthase determines pathogenesis in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence compounds

Chitin, a beta-1,4-linked polysaccharide of N-acetylglucosamine, is a major structural component of fungal cell walls. Fungi have multiple classes of chitin synthases that catalyse N-acetylglucosamine polymerization. Here, we demonstrate the requirement for a class V chitin synthase during host infection by the vascular wilt pathogen Fusarium oxysporum. The chsV gene was identified in an insertional mutagenesis screen for pathogenicity mutants. ChsV has a putative myosin motor and a chitin synthase domain characteristic of class V chitin synthases. The chsV insertional mutant and a gene replacement mutant of F. oxysporum display morphological abnormalities such as hyphal swellings that are indicative of alterations in cell wall structure and can be partially restored by osmotic stabilizer. The mutants are unable to infect and colonize tomato plants or to grow invasively on tomato fruit tissue. They are also hypersensitive to plant antimicrobial defence compounds such as the tomato phytoanticipin alpha-tomatine or H2O2. Reintroduction of a functional chsV copy into the mutant restored the growth phenotype of the wild-type strain. These data suggest that F. oxysporum requires a specific class V chitin synthase for pathogenesis, most probably to protect itself against plant defence mechanisms.     

A polysaccharide deacetylase from Puccinia striiformis f. sp. tritici is an important pathogenicity gene that suppresses plant immunity

The cell wall of filamentous fungi, comprised of chitin, polysaccharide and glycoproteins, maintains the integrity of hyphae and protect them from defence responses by potential host plants. Here, we report that one polysaccharide deacetylase of Puccinia striiformis f. sp. tritici (Pst), Pst_13661, suppresses Bax‐induced cell death in plants and Pst_13661 is highly induced during early stages of the interaction between wheat and Pst. Importantly, the transgenic wheat expressing the RNA interference (RNAi) construct of Pst_13661 exhibits high resistance to major Pst epidemic races CYR31, CYR32 and CYR33 by inhibiting growth and development of Pst, indicating that Pst_13661 is an available pathogenicity factor and is a potential target for generating broad‐spectrum resistance breeding material of wheat. It forms a homo‐polymer and has high affinity for chitin and germ tubes of Pst compared with the control. Besides, Pst_13661 suppresses chitin‐induced plant defence in plants. Hence, we infer that Pst_13661 may modify the fungal cell wall to prevent recognition by apoplastic surveillance systems in plants. This study opens new approaches for developing durable disease‐resistant germplasm by disturbing the growth and development of fungi and develops novel strategies to control crop diseases.     

Cytology of infection of 35S-bean chitinase transgenic canola plants by Rhizoctonia solani: cytochemical aspects of chitin breakdown in vivo

Transgenic canola plants containing high, constitutive levels of bean endochitinase have been shown to be more resistant to infection by the soil-borne pathogen, Rhizoctonia solani, than are wild-type plants that lack the chimeric chitinase gene. To determine whether the resistance of the 35S-chitinase plants to Rhizoctonia infection results from an antimicrobial activity of the bean chitinase in planta, an ultrastructural and cyto-chemical study was performed on infected control and transgenic canola plants. Analysis of root tissues of infected wild-type canola plants revealed that R. solani was capable of extensive tissue colonization including the xylem vessels. Pathogen ingress towards the vascular system was associated with marked host cell wall alterations such as disruption of middle lamella matrices that occurred in advance of fungal penetration. Fungal hyphae colonizing these tissues appeared metabolically active as judged by their typical morphological features and their extensive multiplication. In infected transgenic plants, however, the pattern of fungal colonization was different to that observed in wild-type plants. Penetration of the host cuticle and epidermis was frequently observed, but fungal colonization was usually restricted to the cortex although, in a few cases, some fungal cells could be seen in xylem vessels. In all samples examined, severe hyphal alterations ranging from increased vacuolization to cell lysis were seen. Hyphae occasionally seen in xylem vessels were markedly damaged and often reduced to convoluted wall fragments. Cytochemical labeling of chitin using the WGA/ovomucoid-gold complex showed that hyphal alterations correlated with extensive chitin degradation. Thus, reduction in fungal biomass, increase in hyphal alterations leading to fungal lysis and chitin breakdown appear to be typical features observed in transgenic canola plants. Because these features were not seen in infected wild-type plants, it is likely that constitutive expression of the bean endochitinase gene is, at least in part, responsible for the enhanced protection against fungal attack observed in these plants. It is not known, however, if other components of the host defense response contribute to the resistance phenotype.     

The arms race continues: battle strategies between plants and fungal pathogens

Plants are under constant attack by a vast array of pathogens. To impede their attackers they use both broad-spectrum and pathogen-specific defence mechanisms. The arms race between plants and fungal pathogens is fascinatingly varied, and what might be elicited as a plant defence mechanism against a pathogen could promote or enhance the virulence of other pathogens. Fungi use countermeasures to detoxify plant antimicrobial compounds and to evade host resistance mechanisms. Certain fungal species also manipulate the host hormone balance to create an environment that is beneficial to their survival. Several lines of evidence indicate a co-evolutionary arms race in which both plants and fungi can respond to changes that occur in their opponents.     

The LysM receptor kinase CERK1 mediates bacterial perception in Arabidopsis

Plants use pattern recognition receptors (PRRs) to perceive pathogen-associated molecular pattern (PAMPs) and initiate defence responses. PAMP-triggered immunity (PTI) plays an important role in general resistance, and constrains the growth of most microbes on plants. Despite the importance of PRRs in plant immunity, the vast majority of them remain to be identified. We recently showed that the Arabidopsis LysM receptor kinase CERK1 is required not only for chitin signalling and fungal resistance, but plays an essential role in restricting bacterial growth on plants. We proposed that CERK1 may mediate the perception of a bacterial PAMP, or an endogenous plant cell wall component released during infection, through its extracellular carbohydrate-binding LysM-motifs. Here we report reduced activation of a PAMP-induced defence response on plants lacking the CERK1 gene after treatment with crude bacterial extracts. This demonstrates that CERK1 mediates perception of an unknown bacterial PAMP in Arabidopsis.Key words: PAMP, PRR, PTI, LysM, chitin, bacteria, carbohydrate     

LysM结构域及其与植物-真菌相互作用的关系

在长期的进化过程中,植物与真菌之间形成了复杂而又紧密的联系,其中最主要的就是侵染与防御的关系。植物的抗病性由于涉及农作物、林木的生长与产量,逐渐成为研究热点。在植物免疫系统中,对病原真菌的识别是一个重要环节。目前认为在这一过程中,LysM结构域起到了极为关键的作用。植物细胞膜上有含LysM结构域的识别受体,该受体可以结合真菌细胞壁上的几丁质,并将信号传递到胞内,从而启动免疫反应。在真菌中,同样具有含LysM结构域的基因,主要是一类效应因子。它们可能参与真菌在侵染过程中的"伪装",以逃避植物的识别。该文以LysM结构域在植物-真菌相互作用中扮演的角色为着眼点,讨论有关研究的意义与趋势,并对如何利用LysM结构域的相关研究进行有效的抗病育种提出了新的设想。     

Mining the plant-herbivore interface with a leafmining Drosophila of Arabidopsis

Experimental infections of Arabidopsis thaliana (Arabidopsis) with genomically characterized plant pathogens such as Pseudomonas syringae have facilitated the dissection of canonical eukaryotic defence pathways and parasite virulence factors. Plants are also attacked by herbivorous insects, and the development of an ecologically relevant genetic model herbivore that feeds on Arabidopsis will enable the parallel dissection of host defence and reciprocal resistance pathways such as those involved in xenobiotic metabolism. An ideal candidate is Scaptomyza flava, a drosophilid fly whose leafmining larvae are true herbivores that can be found in nature feeding on Arabidopsis and other crucifers. Here, we describe the life cycle of S.?flava on Arabidopsis and use multiple approaches to characterize the response of Arabidopsis to S.?flava attack. Oviposition choice tests and growth performance assays on different Arabidopsis ecotypes, defence-related mutants, and hormone and chitin-treated plants revealed significant differences in host preference and variation in larval performance across Arabidopsis accessions. The jasmonate and glucosinolate pathways in Arabidopsis are important in mediating quantitative resistance against S.?flava, and priming with jasmonate or chitin resulted in increased resistance. Expression of xenobiotic detoxification genes was reduced in S.?flava larvae reared on Arabidopsis jasmonate signalling mutants and increased in plants pretreated with chitin. These results and future research directions are discussed in the context of developing a genetic model system to analyse insect-plant interactions.     

Irregular growth forms and cell wall modifications,polygalacturonase detection,and endocell formation in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">radicis-lycopersici</Emphasis> infecting tomato plants,as studied ultrastructurally and cytochemically

Pathogen cells of Fusarium oxysporum f.sp. radicis-lycopersici infecting container-grown tomato plants were characterized ultrastructurally, using gold-complexed probes, chitinase and wheat germ agglutinin to localize chitin, and polyclonal antibodies to a polygalacturonase to localize this enzyme. It was isolated and purified from the pathogen growing in culture. Many fungal cells were of irregular forms (microhyphal, frondose) with modified, thin or imperceptible lucent wall layers, in which were often included components seemingly of host origin. Gold particles of the polygalacturonase probe were concentrated on portions of penetration hyphae and in areas of associated altered host wall. Fine filamentous-like structures, often linked to fungal cells, reached into extracellular matter and into host walls. Examination of 0.2–0.25 μm-thick sections at 120 kV, and tilted at various angles, indicated that fungal cells frequently had a pronounced wavy contour. Labelling of thin walls for chitin was mostly nil, particularly in contact with host walls, as of also thicker walls in similar situations, or it was then associated with the outside opaque layer. Cells of diverse dimensions with thin or thicker walls and with altered or normal content, contained endocells. Walls of the encodcells and of the enclosing cells often labelled differently for chitin with both probes. Endocells mostly did not originate from proliferation of a living into a dead cell but often ensuing as an apparent fragmentation of the cell content or following its retraction. The bearing of these observations on the host-pathogen relationship, particularly concerning the role of thin-walled hyphae and irregular forms, is discussed.     

Virus perception at the cell surface: revisiting the roles of receptor-like kinases as viral pattern recognition receptors

Activation of antiviral innate immune responses depends on the recognition of viral components or viral effectors by host receptors. This virus recognition system can activate two layers of host defence, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). While ETI has long been recognized as an efficient plant defence against viruses, the concept of antiviral PTI has only recently been integrated into virus–host interaction models, such as the RNA silencing-based defences that are triggered by viral dsRNA PAMPs produced during infection. Emerging evidence in the literature has included the classical PTI in the antiviral innate immune arsenal of plant cells. Therefore, our understanding of PAMPs has expanded to include not only classical PAMPS, such as bacterial flagellin or fungal chitin, but also virus-derived nucleic acids that may also activate PAMP recognition receptors like the well-documented phenomenon observed for mammalian viruses. In this review, we discuss the notion that plant viruses can activate classical PTI, leading to both unique antiviral responses and conserved antipathogen responses. We also present evidence that virus-derived nucleic acid PAMPs may elicit the NUCLEAR SHUTTLE PROTEIN-INTERACTING KINASE 1 (NIK1)-mediated antiviral signalling pathway that transduces an antiviral signal to suppress global host translation.     

Cleavage of chitinous elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme by host chitinases prevents induction of K+ and Cl− release, extracellular alkalinization and H2O2 synthesis of Picea abies cells

Rapid reactions comprising efflux of K⁺ and Cl⁻, phosphorylation of a 63-kDa protein (pp63), extracellular alkalinization and synthesis of H₂O₂ are equally induced in cells of Picea abies (L.) Karst. by chitotetraose, colloidal chitin and cell wall elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. an ectomycorrhizal partner of spruce. Cleavage of fungal cell wall elicitors and of artificial chitin elicitors to monomeric and dimeric fragments by apoplasmic spruce chitinases (36-kDa class I chitinase, pI 8.0, and 28-kDa chitinase, pI 8.7; EC 3.2.1.14) equally prevented induction of these rapid reactions. Also, N-acetylglucosamine oligomers and elicitors from the fungal cell walls showed a similar dependence of their activity on the degree of polymerisation. From these results it is suggested that, during ectomycorrhiza formation, only some of the chitin-derived elicitors reach their receptors at the plant plasma membrane, initiating reactions of the hypersensitive response in the host cells. The remaining fungal elicitors will be degraded to varying extents by wall-localized chitinases of the host root, reducing the defence reactions of the plant and allowing symbiotic interactions of both organisms. Received: 6 January 1997 / Accepted: 14 March 1997     

A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis

Chitin, a polymer of N-acetyl-d-glucosamine, is found in fungal cell walls but not in plants. Plant cells can perceive chitin fragments (chitooligosaccharides) leading to gene induction and defense responses. We identified a LysM receptor-like protein (LysM RLK1) required for chitin signaling in Arabidopsis thaliana. The mutation in this gene blocked the induction of almost all chitooligosaccharide-responsive genes and led to more susceptibility to fungal pathogens but had no effect on infection by a bacterial pathogen. Additionally, exogenously applied chitooligosaccharides enhanced resistance against both fungal and bacterial pathogens in the wild-type plants but not in the mutant. Together, our data indicate that LysM RLK1 is essential for chitin signaling in plants (likely as part of the receptor complex) and is involved in chitin-mediated plant innate immunity. The LysM RLK1-mediated chitin signaling pathway is unique, but it may share a conserved downstream pathway with the FLS2/flagellin- and EFR/EF-Tu-mediated signaling pathways. Additionally, our work suggests a possible evolutionary relationship between the chitin and Nod factor perception mechanisms due to the similarities between their potential receptors and between the signal molecules perceived by them.     

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.     

Ergosterol,an orphan fungal microbe‐associated molecular pattern (MAMP)

Fungal pathogens continue to pose a significant threat to crop production and food supply. The early stages of plant–fungus interactions are mostly mediated by microbe‐associated molecular pattern (MAMP) molecules, perceived by plant pattern recognition receptors (PRRs). Currently, the identified fungal MAMP molecules include chitin, chitosan, β‐glucans, elicitins and ergosterol. Although the molecular battles between host plants and infecting fungal phytopathogens have been studied extensively, many aspects still need to be investigated to obtain a holistic understanding of the intrinsic mechanisms, which is paramount in combating fungal plant diseases. Here, an overview is given of the most recent findings concerning an ‘orphan’ fungal MAMP molecule, ergosterol, and we present what is currently known from a synopsis of different genes, proteins and metabolites found to play key roles in induced immune responses in plant–fungus interactions. Clearly, integrative investigations are still needed to provide a comprehensive systems‐based understanding of the dynamics associated with molecular mechanisms in plant–ergosterol interactions and associated host responses.     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

NPR1 and EDS11 contribute to host resistance against Fusarium culmorum in Arabidopsis buds and flowers

The cereal ear blight fungal pathogen Fusarium culmorum can infect Arabidopsis floral tissue, causing disease symptoms and mycotoxin production. Here we assessed the effect of seven mutants and one transgenic overexpression line, residing in either the salicylic acid (SA), jasmonic acid (JA) or ethylene (ET) defence signalling pathways, on the outcome of the Fusarium –Arabidopsis floral interaction. The bacterial susceptiblity mutant eds11 was also assessed. Flowering plants were spray inoculated with F. culmorum conidia to determine the host responses to initial infection and subsequent colonization. Enhanced susceptibility and higher concentrations of deoxynivalenol mycotoxin were observed in buds and flowers of the npr1 and eds11 mutants than in the wild-type Col-0 plants. An effect of the other two defence signalling pathways on disease was either absent (ET/JA combined), absent/minimal (ET) or inconclusive (JA). Overall, this study highlights a role for NPR1 and EDS11 in basal defence against F. culmorum in some floral organs. This is the first time that any of these well-characterized defence signalling mutations have been evaluated for a role in floral defence in any plant species.     

Chitin-supplemented Foliar Application of Serratia marcescens GPS 5 Improves Control of Late Leaf Spot Disease of Groundnut by Activating Defence-related Enzymes

Chitinolytic Serratia marcescens GPS 5 and non‐chitinolytic Pseudomonas aeruginosa GSE 18, with and without supplementation of chitin, were tested for their ability to activate defence‐related enzymes in groundnut leaves. Thirty‐day‐old groundnut (cv. TMV 2) plants pretreated with GPS 5 and GSE 18 (with and without supplementation of 1% colloidal chitin) were challenge inoculated after 24 h with Phaeoisariopsis personata, the causal agent of late leaf spot (LLS) disease of groundnut. GPS 5 and GSE 18, applied as a prophylactic spray, reduced the lesion frequency by 23% and 67%, respectively, compared with control. Chitin supplementation had no effect on the control of LLS by GSE 18, unlike GPS 5, which upon chitin supplementation reduced the lesion frequency by 64%, compared with chitin alone. In a time course study the activities of chitinase, β‐1,3‐glucanase, peroxidase and phenylalanine ammonia lyase were determined for the different treatments. There was an enhanced activity of the four defence‐related enzymes with all the bacterial treatments when compared with phosphate buffer and colloidal chitin‐treated controls. In correlation to disease severity in bacterial treatments, chitin‐supplemented GSE 18 was similar to GSE 18, whereas chitin‐supplemented GPS 5 was much more effective than GPS 5, in activation of the defence‐related enzymes. The high levels of enzyme activities following chitin‐supplemented GPS 5 application continued up to the measured 13 days after pathogen inoculation.