Large‐scale molecular genetic analysis in plant‐pathogenic fungi: a decade of genome‐wide functional analysis

Plant‐pathogenic fungi cause diseases to all major crop plants world‐wide and threaten global food security. Underpinning fungal diseases are virulence genes facilitating plant host colonization that often marks pathogenesis and crop failures, as well as an increase in staple food prices. Fungal molecular genetics is therefore the cornerstone to the sustainable prevention of disease outbreaks. Pathogenicity studies using mutant collections provide immense function‐based information regarding virulence genes of economically relevant fungi. These collections are rich in potential targets for existing and new biological control agents. They contribute to host resistance breeding against fungal pathogens and are instrumental in searching for novel resistance genes through the identification of fungal effectors. Therefore, functional analyses of mutant collections propel gene discovery and characterization, and may be incorporated into disease management strategies. In the light of these attributes, mutant collections enhance the development of practical solutions to confront modern agricultural constraints. Here, a critical review of mutant collections constructed by various laboratories during the past decade is provided. We used Magnaporthe oryzae and Fusarium graminearum studies to show how mutant screens contribute to bridge existing knowledge gaps in pathogenicity and fungal–host interactions.     

Characterization of protein kinase PsSRPKL,a novel pathogenicity factor in the wheat stripe rust fungus

As in other eukaryotes, protein kinases (PKs) are generally evolutionarily conserved and play major regulatory roles in plant pathogenic fungi. Many PKs have been proven to be important for pathogenesis in model fungal plant pathogens, but little is currently known about their roles in the pathogenesis of cereal rust fungi, devastating pathogens in agriculture worldwide. Here, we report on an in planta highly induced PK gene PsSRPKL from the wheat stripe rust fungus Puccinia striiformis f. sp. tritici (Pst), one of the most important cereal rust fungi. PsSRPKL belongs to a group of PKs that are evolutionarily specific to cereal rust fungi. It shows a high level of intraspecies polymorphism in the kinase domains and directed green fluorescent protein chimers to plant nuclei. Overexpression of PsSRPKL in fission yeast induces aberrant cell morphology and a decreased resistance to environmental stresses. Most importantly, PsSRPKL is proven to be an important pathogenicity factor responsible for fungal growth and responses to environmental stresses, therefore contributing significantly to Pst virulence in wheat. We hypothesize that cereal rust fungi have developed specific PKs as pathogenicity factors for adaptation to their host species during evolution. Thus, our findings provide significant insights into pathogenicity and virulence evolution in cereal rust fungi.     

Host‐induced gene silencing of an important pathogenicity factor PsCPK1 in Puccinia striiformis f. sp. tritici enhances resistance of wheat to stripe rust

Rust fungi are devastating plant pathogens and cause a large economic impact on wheat production worldwide. To overcome this rapid loss of resistance in varieties, we generated stable transgenic wheat plants expressing short interfering RNAs (siRNAs) targeting potentially vital genes of Puccinia striiformis f. sp. tritici (Pst). Protein kinase A (PKA) has been proved to play important roles in regulating the virulence of phytopathogenic fungi. PsCPK1, a PKA catalytic subunit gene from Pst, is highly induced at the early infection stage of Pst. The instantaneous silencing of PsCPK1 by barley stripe mosaic virus (BSMV)‐mediated host‐induced gene silencing (HIGS) results in a significant reduction in the length of infection hyphae and disease phenotype. These results indicate that PsCPK1 is an important pathogenicity factor by regulating Pst growth and development. Two transgenic lines expressing the RNA interference (RNAi) construct in a normally susceptible wheat cultivar displayed high levels of stable and consistent resistance to Pst throughout the T₃ to T₄ generations. The presence of the interfering RNAs in transgenic wheat plants was confirmed by northern blotting, and these RNAs were found to efficiently down‐regulate PsCPK1 expression in wheat. This study addresses important aspects for the development of fungal‐derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control cereal rust diseases.     

PEX基因在过氧化物酶体形成及真菌致病性中的作用

过氧化物酶体(peroxisomes)是一类真核生物中普遍存在的细胞器,参与β-氧化、乙醛酸循环等多种重要的生化代谢。研究表明,过氧化物酶体在植物病原真菌侵染寄主过程中具有着举足轻重的作用。参与过氧化物酶体形成与增殖的基因,通常称为PEX基因。近年来,越来越多的PEX基因在植物病原真菌中得到鉴定,真菌过氧化物酶体的形成机制及其在植物病原真菌生长发育和致病过程中的作用越来越受到研究者的关注。本文围绕PEX 基因在过氧化物酶体形成中的作用、对过氧化物酶体相关生化代谢的影响,以及与植物病原真菌生长发育和致病性的关系进行了综述,以期为植物病原真菌致病机理研究和病害防控提供借鉴和参考。     

Melanization of appressoria is critical for the pathogenicity of <Emphasis Type="Italic">Diplocarpon rosae</Emphasis>

Previous studies have shown the role of melanized appressoria in the pathogenicity of various fungi. Diplocarpon rosae is a worldwide outdoor fungal pathogen of rose plants causing black spot disease of rose leaves. To fully understand how this fungus colonizes its host, which is critical for the development of an efficient and sustainable disease management program, we studied the fungal (especially the appressoria) structures of D. rosae in detail at an early stage of infection. Using both microscopic and biochemical analyses, we observed strong melanized appressoria formation localized at the point of D. rosae penetration, which forms the pathogen–plant interface. Treatment of infected plants with melanin biosynthesis inhibitors (MBIs) prevented melanization of D. rosae appressoria and positively correlated with significant reductions in black spot disease symptoms, suggesting that melanization of appressoria might be a critical factor for the pathogenicity of D. rosae. Our findings were confirmed and validated by the lack of melanized appressorial ring formation on an artificial surface and on a D. rosae-non host plant system, Arabidopsis thaliana. Our findings suggest that localized melanization of appressoria is a crucial factor for the pathogenicity of D. rosae and treatment of the fungus with MBIs seems to be a promising disease management alternative for black spot disease of roses.     

Fungal resistance to plant antibiotics as a mechanism of pathogenesis.

Many plants produce low-molecular-weight compounds which inhibit the growth of phytopathogenic fungi in vitro. These compounds may be preformed inhibitors that are present constitutively in healthy plants (also known as phytoanticipins), or they may be synthesized in response to pathogen attack (phytoalexins). Successful pathogens must be able to circumvent or overcome these antifungal defenses, and this review focuses on the significance of fungal resistance to plant antibiotics as a mechanism of pathogenesis. There is increasing evidence that resistance of fungal pathogens to plant antibiotics can be important for pathogenicity, at least for some fungus-plant interactions. This evidence has emerged largely from studies of fungal degradative enzymes and also from experiments in which plants with altered levels of antifungal secondary metabolites were generated. Whereas the emphasis to date has been on degradative mechanisms of resistance of phytopathogenic fungi to antifungal secondary metabolites, in the future we are likely to see a rapid expansion in our knowledge of alternative mechanisms of resistance. These may include membrane efflux systems of the kind associated with multidrug resistance and innate resistance due to insensitivity of the target site. The manipulation of plant biosynthetic pathways to give altered antibiotic profiles will also be valuable in telling us more about the significance of antifungal secondary metabolites for plant defense and clearly has great potential for enhancing disease resistance for commercial purposes.     

Fungal Resistance to Plant Antibiotics as a Mechanism of Pathogenesis

Many plants produce low-molecular-weight compounds which inhibit the growth of phytopathogenic fungi in vitro. These compounds may be preformed inhibitors that are present constitutively in healthy plants (also known as phytoanticipins), or they may be synthesized in response to pathogen attack (phytoalexins). Successful pathogens must be able to circumvent or overcome these antifungal defenses, and this review focuses on the significance of fungal resistance to plant antibiotics as a mechanism of pathogenesis. There is increasing evidence that resistance of fungal pathogens to plant antibiotics can be important for pathogenicity, at least for some fungus-plant interactions. This evidence has emerged largely from studies of fungal degradative enzymes and also from experiments in which plants with altered levels of antifungal secondary metabolites were generated. Whereas the emphasis to date has been on degradative mechanisms of resistance of phytopathogenic fungi to antifungal secondary metabolites, in the future we are likely to see a rapid expansion in our knowledge of alternative mechanisms of resistance. These may include membrane efflux systems of the kind associated with multidrug resistance and innate resistance due to insensitivity of the target site. The manipulation of plant biosynthetic pathways to give altered antibiotic profiles will also be valuable in telling us more about the significance of antifungal secondary metabolites for plant defense and clearly has great potential for enhancing disease resistance for commercial purposes.     

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.     

Plant phosphatidylinositol‐specific phospholipase C at the center of plant innate immunity

Understanding plant resistance to pathogenic microbes requires detailed information on the molecular mechanisms controlling the execution of plant innate immune responses. A growing body of evidence places phosphoinositide‐specific phospholipase C (PI‐PLC) enzymes immediately downstream of activated immune receptors, well upstream of the initiation of early defense responses. An increase of the cytoplasmic levels of free Ca²⁺, lowering of the intercellular pH and the oxidative burst are a few examples of such responses and these are regulated by PI‐PLCs. Consequently, PI‐PLC activation represents an early primary signaling switch between elicitation and response involving the controlled hydrolysis of essential signaling phospholipids, thereby simultaneously generating lipid and non‐lipid second messenger molecules required for a swift cellular defense response. Here, we elaborate on the signals generated by PI‐PLCs and their respective downstream effects, while providing an inventory of different types of evidence describing the involvement of PI‐PLCs in various aspects of plant immunity. We project the discussed information into a model describing the cellular events occurring after the activation of plant immune receptors. With this review we aim to provide new insights supporting future research on plant PI‐PLCs and the development of plants with improved resistance.     

Interactions between functionally diverse fungal mutualists inconsistently affect plant performance and competition

Plants form mutualistic relationship with a variety of belowground fungal species. Such a mutualistic relationship can enhance plant growth and resistance to pathogens. Yet, we know little about how interactions between functionally diverse groups of fungal mutualists affect plant performance and competition. We experimentally determined the effects of interaction between two functional groups of belowground fungi that form mutualistic relationship with plants, arbuscular mycorrhizal (AM) fungi and Trichoderma, on interspecific competition between pairs of closely related plant species from four different genera. We hypothesized that the combination of two functionally diverse belowground fungal species would allow plants and fungi to partition their symbiotic relationships and relax plant–plant competition. Our results show that: 1) the AM fungal species consistently outcompeted the Trichoderma species independent of plant combinations; 2) the fungal species generally had limited effects on competitive interactions between plants; 3) however, the combination of fungal species relaxed interspecific competition in one of the four instances of plant–plant competition, despite the general competitive superiority of AM fungi over Trichoderma. We highlight that the competitive outcome between functionally diverse fungal species may show high consistency across a broad range of host plants and their combinations. However, despite this consistent competitive hierarchy, the consequences of their interaction for plant performance and competition can strongly vary among plant communities.     

Untangling above‐ and belowground mycorrhizal fungal networks in tropical orchids

Orchids typically depend on fungi for establishment from seeds, forming mycorrhizal associations with basidiomycete fungal partners in the polyphyletic group rhizoctonia from early stages of germination, sometimes with very high specificity. This has raised important questions about the roles of plant and fungal phylogenetics, and their habitat preferences, in controlling which fungi associate with which plants. In this issue of Molecular Ecology, Martos et al. (2012) report the largest network analysis to date for orchids and their mycorrhizal fungi, sampling a total of over 450 plants from nearly half the 150 tropical orchid species on Reunion Island, encompassing its main terrestrial and epiphytic orchid genera. The authors found a total of 95 operational taxonomic units of mycorrhizal fungi and investigated the architecture and nestedness of their bipartite networks with 73 orchid species. The most striking finding was a major ecological barrier between above‐ and belowground mycorrhizal fungal networks, despite both epiphytic and terrestrial orchids often associating with closely related taxa across all three major lineages of rhizoctonia fungi. The fungal partnerships of the epiphytes and terrestrial species involved a diversity of fungal taxa in a modular network architecture, with only about one in ten mycorrhizal fungi partnering orchids in both groups. In contrast, plant and fungal phylogenetics had weak or no effects on the network. This highlights the power of recently developed ecological network analyses to give new insights into controls on plant–fungal symbioses and raises exciting new hypotheses about the differences in properties and functioning of mycorrhiza in epiphytic and terrestrial orchids.