The lifestyle transition of Arthrobotrys oligospora is mediated by microRNA-like RNAs

The lifestyle transition of fungi, defined as switching from taking organic material as nutrients to pathogens, is a fundamental phenomenon in nature. However, the mechanisms of such transition remain largely unknown. Here we show microRNA-like RNAs(milRNAs) play a key role in fungal lifestyle transition for the first time. We identified milRNAs by small RNA sequencing in Arthrobotrys oligospora, a known nematode-trapping fungus. Among them, 7 highly expressed milRNAs were confirmed by northern-blot analysis. Knocking out two milRNAs significantly decreased A. oligospora's ability to switch lifestyles. We further identified that two of these milRNAs were associated with argonaute protein QDE-2 by RNA-immunoprecipitation(RIP) analysis. Three of the predicted target genes of milRNAs were found in immunoprecipitation(IP)products of QDE-2. Disruption of argonaute gene qde-2 also led to serious defects in lifestyle transition. Interestingly, knocking out individual milRNAs or qde-2 lead to diverse responses under different conditions, and qde-2 itself may be targeted by the milRNAs. Collectively, it indicates the lifestyle transition of fungi is mediated by milRNAs through RNA interference(RNAi)machinery, revealing the wide existence of miRNAs in fungi kingdom and providing new insights into understanding the adaptation of fungi from scavengers to predators and the mechanisms underlying fungal infections.     

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.     

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.     

Powdery mildew susceptibility and biotrophic infection strategies

Plants are resistant to most potentially pathogenic microbes. This forces plant pathogens to develop sophisticated strategies to overcome basic plant resistance, either by masking intrusion or by suppression of host defences. This is particularly true for fungal pathogens, which establish long lasting interactions with living host tissue, without causing visible damage to invaded cells. The interactions of cereal crops and Arabidopsis with powdery mildew fungi are model systems for understanding host resistance. Currently, these systems are also promoting the understanding of fungal infection by identifying fungal pathogenicity and virulence factors and host target sites. This minireview focuses on recent findings about host susceptibility and the way powdery mildew fungi might induce it.     

Virulence genes and the evolution of host specificity in plant-pathogenic fungi

In the fungal kingdom, the ability to cause disease in plants appears to have arisen multiple times during evolution. In many cases, the ability to infect particular plant species depends on specific genes that distinguish virulent fungi from their sometimes closely related nonvirulent relatives. These genes encode host-determining "virulence factors," including small, secreted proteins and enzymes involved in the synthesis of toxins. These virulence factors typically are involved in evolutionary arms races between plants and pathogens. We briefly summarize current knowledge of these virulence factors from several fungal species in terms of function, phylogenetic distribution, sequence variation, and genomic location. Second, we address some issues that are relevant to the evolution of virulence in fungi toward plants; in particular, horizontal gene transfer and the genomic organization of virulence genes.     

Fungal effector proteins: past, present and future

The pioneering research of Harold Flor on flax and the flax rust fungus culminated in his gene-for-gene hypothesis. It took nearly 50 years before the first fungal avirulence ( Avr ) gene in support of his hypothesis was cloned. Initially, fungal Avr genes were identified by reverse genetics and map-based cloning from model organisms, but, currently, the availability of many sequenced fungal genomes allows their cloning from additional fungi by a combination of comparative and functional genomics. It is believed that most Avr genes encode effectors that facilitate virulence by suppressing pathogen-associated molecular pattern-triggered immunity and induce effector-triggered immunity in plants containing cognate resistance proteins. In resistant plants, effectors are directly or indirectly recognized by cognate resistance proteins that reside either on the plasma membrane or inside the plant cell. Indirect recognition of an effector (also known as the guard model) implies that the virulence target of an effector in the host (the guardee) is guarded by the resistance protein (the guard) that senses manipulation of the guardee, leading to activation of effector-triggered immunity. In this article, we review the literature on fungal effectors and some pathogen-associated molecular patterns, including those of some fungi for which no gene-for-gene relationship has been established.     

FoQDE2-dependent milRNA promotes Fusarium oxysporum f. sp. cubense virulence by silencing a glycosyl hydrolase coding gene expression

MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein-coding gene expression primarily found in plants and animals. Fungi produce microRNA-like RNAs (milRNAs) that are structurally similar to miRNAs and functionally important in various biological processes. The fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Banana Fusarium vascular wilt that threatens global banana production. It remains uncharacterized about the biosynthesis and functions of milRNAs in Foc. In this study, we investigated the biological function of milRNAs contributing to Foc pathogenesis. Within 24 hours post infecting the host, the Argonaute coding gene FoQDE2, and two Dicer coding genes FoDCL1 and FoDCL2, all of which are involved in milRNA biosynthesis, were significantly induced. FoQDE2 deletion mutant exhibited decreased virulence, suggesting the involvement of milRNA biosynthesis in the Foc pathogenesis. By small RNA sequencing, we identified 364 small RNA-producing loci in the Foc genome, 25 of which were significantly down-regulated in the FoQDE2 deletion mutant, from which milR-87 was verified as a FoQDE2-depedent milRNA based on qRT-PCR and Northern blot analysis. Compared to the wild-type, the deletion mutant of milR-87 was significantly reduced in virulence, while overexpression of milR-87 enhanced disease severity, confirming that milR-87 is crucial for Foc virulence in the infection process. We furthermore identified FOIG_15013 (a glycosyl hydrolase-coding gene) as the direct target of milR-87 based on the expression of FOIG_15013-GFP fusion protein. The FOIG_15013 deletion mutant displayed similar phenotypes as the overexpression of milR-87, with a dramatic increase in the growth, conidiation and virulence. Transient expression of FOIG_15013 in Nicotiana benthamiana leaves activates the host defense responses. Collectively, this study documents the involvement of milRNAs in the manifestation of the devastating fungal disease in banana, and demonstrates the importance of milRNAs in the pathogenesis and other biological processes. Further analyses of the biosynthesis and expression regulation of fungal milRNAs may offer a novel strategy to combat devastating fungal diseases.     

Signalling between Pathogenic Rust Fungi and Resistant or Susceptible Host Plants

Rust fungi are obligately biotrophic plant parasites that obtaintheir nutrients from living host cells. The initiation of thetwo parasitic phases of these fungi generally requires topographicsignals from the plant surface followed, for the dikaryoticphase, by a successive sequence of signals to control furtherfungal development within the plant. During the fungal lifecycle, three types of intracellular structures (invasion hyphae,M-, and D-haustoria) are formed and each may differently affectthe host membrane that surrounds it, as well as affecting othercellular components. Each intracellular structure also preventsnon-specific plant defences triggered by fungal activities,possibly by interfering with the signalling system rather thandefence expression. In resistant host cultivars, cellular invasiontriggers a rapid cell death (the hypersensitive response) thatshares some features with developmentally programmed cell deathin animal and plant tissues, and is controlled by parasite-specificresistance genes that resemble those that defend plants againstother types of pathogens. Evidence from one system suggeststhat this response is specifically elicited by a fungal peptideand does not involve the oxidative burst typical of resistanceexpression in other plant-pathogen interactions. However, overall,few of the molecules involved in any of these plant-rust fungiinteractions have been completely characterized and much isleft to be discovered, particularly with respect to how cellularsusceptibility to rust fungi is conditioned.Copyright 1997 Annalsof Botany Company Apoptosis; biotrophy; elicitor; hypersensitive response; oxidative burst; suppressor; Uromyces vignae     

Interspecific hybridization in plant-associated fungi and oomycetes: a review

Fungi (kingdom Mycota) and oomycetes (kingdom Stramenopila, phylum Oomycota) are crucially important in the nutrient cycles of the world. Their interactions with plants sometimes benefit and sometimes act to the detriment of humans. Many fungi establish ecologically vital mutualisms, such as in mycorrhizal fungi that enhance nutrient acquisition, and endophytes that combat insects and other herbivores. Other fungi and many oomycetes are plant pathogens that devastate natural and agricultural populations of plant species. Studies of fungal and oomycete evolution were extraordinarily difficult until the advent of molecular phylogenetics. Over the past decade, researchers applying these new tools to fungi and oomycetes have made astounding new discoveries, among which is the potential for interspecific hybridization. Consequences of hybridization among pathogens include adaptation to new niches such as new host species, and increased or decreased virulence. Hybrid mutualists may also be better adapted to new hosts and can provide greater or more diverse benefits to host plants.     

Horizontal gene and chromosome transfer in plant pathogenic fungi affecting host range

Plant pathogenic fungi adapt quickly to changing environments including overcoming plant disease resistance genes. This is usually achieved by mutations in single effector genes of the pathogens, enabling them to avoid recognition by the host plant. In addition, horizontal gene transfer (HGT) and horizontal chromosome transfer (HCT) provide a means for pathogens to broaden their host range. Recently, several reports have appeared in the literature on HGT, HCT and hybridization between plant pathogenic fungi that affect their host range, including species of Stagonospora/Pyrenophora, Fusarium and Alternaria. Evidence is given that HGT of the ToxA gene from Stagonospora nodorum to Pyrenophora tritici-repentis enabled the latter fungus to cause a serious disease in wheat. A nonpathogenic Fusarium species can become pathogenic on tomato by HCT of a pathogenicity chromosome from Fusarium oxysporum f.sp lycopersici, a well-known pathogen of tomato. Similarly, Alternaria species can broaden their host range by HCT of a single chromosome carrying a cluster of genes encoding host-specific toxins that enabled them to become pathogenic on new hosts such as apple, Japanese pear, strawberry and tomato, respectively. The mechanisms HGT and HCT and their impact on potential emergence of fungal plant pathogens adapted to new host plants will be discussed.     

Groovy times: filamentous pathogen effectors revealed

Filamentous microorganisms, such as fungi and oomycetes, secrete an arsenal of effector proteins that modulate plant innate immunity and enable parasitic infection. Deciphering the biochemical activities of effectors to understand how pathogens successfully colonize and reproduce on their host plants became a driving paradigm in the field of fungal and oomycete pathology. Recent findings illustrate a diversity of effector structures and activities, as well as validate the view that effector genes are the target of the evolutionary forces that drive the antagonistic interplay between pathogen and host.     

A Dual Role for Microbial Pathogen-Derived Effector Proteins in Plant Disease and Resistance

Many proteins from plant pathogens affecting the interaction with the host plant have dual functions: they promote virulence on the host species and they function as avirulence determinants by eliciting defense reactions in host cultivars expressing the appropriate resistance genes. In viruses all proteins encoded by the small genomes can be expected to be essential for viral development in the host. However, in different plants surveillance systems have evolved that are able to recognize most of these proteins. Bacteria and fungi have specialized pathogenicity and virulence genes. Many of the latter were originally identified through the resistance gene-dependent elicitor activity of their products. Their role in virulence only became apparent when they were inactivated or transferred to different microbes or after their ectopic expression in host plants. Many microbes appear to maintain these genes despite their disadvantageous effect, introducing only few mutations to abolish the interaction of their products with the plant recognition system. This has been interpreted as been indicative of a virulence function of the gene products that is not impaired by the mutations. Alternatively, in particular in bacteria there is now evidence that pathogenicity was acquired through horizontal gene transfer. Genes supporting virulence in the donor organism's original host appear to have traveled along. Being gratuitous in the new situation, they may have been inactivated without loss of any beneficial function for the pathogen.     

Coevolutionary genetics of plants and pathogens

Summary The genetic polymorphism maintained by host-pathogen coevolution is analysed in a multilocus model. The model assumes gene-for-gene interactions of the type commonly observed between host plants and their fungal pathogens. Unstable (epidemic) systems maintain more resistance genes, fewer virulence genes, and less overall genetic diversity than stable (endemic) diseases. The stability of the system depends primarily on demographic parameters, such as the pathogen's intrinsic rate of increase, rather than genetic parameters, such as the costs of resistance and virulence. At equilibrium the model predicts that the number of resistance alleles in each host plant follows a binomial distribution that depends on the cost to the pathogen for carrying virulence alleles. Similarly, the number of virulence alleles in each pathogen spore follows a binomial distribution that depends on one minus the cost to the host for carrying resistance alleles. Data from wild populations match the predicted binomial distributions.     

The essential role of fungal peroxisomes in plant infection

Several filamentous fungi are ecologically and economically important plant pathogens that infect a broad variety of crops. They cause high annual yield losses and contaminate seeds and fruits with mycotoxins. Not only powerful infection structures and detrimental toxins, but also cell organelles, such as peroxisomes, play important roles in plant infection. In this review, we summarize recent research results that revealed novel peroxisomal functions of filamentous fungi and highlight the importance of peroxisomes for infection of host plants. Central for fungal virulence are two primary metabolic pathways, fatty acid β-oxidation and the glyoxylate cycle, both of which are required to produce energy, acetyl-CoA, and carbohydrates. These are ultimately needed for the synthesis of cell wall polymers and for turgor generation in infection structures. Most novel results stem from different routes of secondary metabolism and demonstrate that peroxisomes produce important precursors and house various enzymes needed for toxin production and melanization of appressoria. All these peroxisomal functions in fungal virulence might represent elegant targets for improved crop protection.     

Plant resistance signalling hijacked by a necrotrophic fungal pathogen

The strategies used by necrotrophic fungal pathogens to infect plants are often perceived as lacking the sophistication of their haustorium producing, host defence suppressing, biotrophic counterparts. There is also a relative paucity of knowledge regarding how effective gene-for-gene based resistance reactions might function against necrotrophic plant pathogens. However, recent data has emerged from a number of systems which has highlighted that particular species of necrotrophic (and/or hemibiotrophic) fungi, have evolved very sophisticated strategies for plant infection which appear, in fact, to hijack the host resistance responses that are commonly deployed against biotrophs. Both disease resistance (R) protein homologues and mitogen-activated protein kinase (MAPK) cascades commonly associated with incompatible disease resistance responses; appear to be targeted by necrotrophic fungi during compatible disease interactions. These findings highlight an emerging sophistication in the strategies deployed by necrotrophic fungi to infect plants.Key words: Mycosphaerella graminicola, Septoria tritici, Triticum aestivum, mitogen-activated protein kinase, programmed cell death, fungal pathogen, disease resistance, disease susceptibility, toxin