The Role of Glycoside Hydrolases in Phytopathogenic Fungi and Oomycetes Virulence

Phytopathogenic fungi need to secrete different hydrolytic enzymes to break down complex polysaccharides in the plant cell wall in order to enter the host and develop the disease. Fungi produce various types of cell wall degrading enzymes (CWDEs) during infection. Most of the characterized CWDEs belong to glycoside hydrolases (GHs). These enzymes hydrolyze glycosidic bonds and have been identified in many fungal species sequenced to date. Many studies have shown that CWDEs belong to several GH families and play significant roles in the invasion and pathogenicity of fungi and oomycetes during infection on the plant host, but their mode of function in virulence is not yet fully understood. Moreover, some of the CWDEs that belong to different GH families act as pathogen-associated molecular patterns (PAMPs), which trigger plant immune responses. In this review, we summarize the most important GHs that have been described in eukaryotic phytopathogens and are involved in the establishment of a successful infection

Taxol and beta-tubulins from endophytic fungi isolated from the Himalayan Yew, Taxus wallichiana Zucc.

Paclitaxel, better known as the anticancer drug Taxol (R), has been isolated from several plant species and has been shown to be produced by fungi, actinomycetes, and even bacteria isolated from marine macroalgae. Given its cytostatic effect, studies conducted in the 1990's showed that paclitaxel was toxic to many pathogenic fungi and oomycetes. Further studies led to the idea that the differences in paclitaxel sensitivity exhibited by different fungi were due to differences in the p-tubulin protein sequence. With the recent isolation of endophytic fungi from the leaves and bark of the Himalayan Yew, Taxus wallichiana Zucc., and the availability of genomes from paclitaxel-producing fungi, we decided to further explore the idea that endophytic fungi isolated from Yews should be well-adapted to their environment by encoding13-tubulin proteins that are insensitive to paclitaxel. Our results found evidence of episodic positive/diversifying selection at 10 sites (default p-value threshold of 0.1) in the 1-tubulin sequences, corresponding to codon positions 33, 55, 172, 218, 279, 335, 359, 362, 379, and 406. Four of these positions (i.e., 172, 279, 359, and 362) have been implicated in the binding of paclitaxel by beta-tubulin or formed part of the binding pocket. As expected, all the fungal endophytes grew in different media regardless of the paclitaxel concentration tested. Furthermore, our results also showed that Taxomyces andreanae CBS 279.92, the first fungus shown to produce paclitaxel, is a Basidiomycete fungus as the two beta tubulins encoded by the fungus clustered together with other Basidiomycete fungi

Investigating the role of a putative endolysin-like candidate effector protein in Verticillium longisporum virulence

Verticillium is a genus of ascomycete fungi that encompasses several plant pathogenic species, and cause severe annual yield losses on many economically important crops worldwide. One of the most important species in this genus, is V. longisporum, which causes disease mainly on plants in the Brassicaceae family. Genome analysis of V. longisporum strain VL1 revealed a number of candidate effector genes that may be associated with fungal virulence. One of these candidate effector-genes encodes a putative endolysin-like protein. Endolysins are hydrolytic enzymes that are secreted by bacteriophages and recently, they have been identified in fungal genomes as well. In this study, the potential role of this gene has been investigated in V. longisporum. Our data showed that this gene was highly induced in the fungus during Brassica napus infection and its overexpression significantly increased V. longisporum virulence, indi-cating an involvement in the fungal infection process.(c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Verticilium longisporum phospholipase VlsPLA(2) is a virulence factor that targets host nuclei and modulates plant immunity

Phospholipase A(2) (PLA(2)) is a lipolytic enzyme that hydrolyses phospholipids in the cell membrane. In the present study, we investigated the role of secreted PLA(2) (VlsPLA(2)) in Verticillium longisporum, a fungal phytopathogen that mostly infects plants belonging to the Brassicaceae family, causing severe annual yield loss worldwide. Expression of the VlsPLA(2) gene, which encodes active PLA(2), is highly induced during the interaction of the fungus with the host plant Brassica napus. Heterologous expression of VlsPLA(2) in Nicotiana benthamiana resulted in increased synthesis of certain phospholipids compared to plants in which enzymatically inactive PLA(2) was expressed (VlsPLA(2)(Delta CD)). Moreover, VlsPLA(2) suppresses the hypersensitive response triggered by the Cf4/Avr4 complex, thereby suppressing the chitin--induced reactive oxygen species burst. VlsPLA(2)-overexpressing V. longisporum strains showed increased virulence in Arabidopsis plants, and transcriptomic analysis of this fungal strain revealed that the induction of the gene contributed to increased virulence. VlsPLA(2) was initially localized to the host nucleus and then translocated to the chloroplasts at later time points. In addition, VlsPLA(2) bound to the vesicle-associated membrane protein A (VAMPA) and was transported to the nuclear membrane. In the nucleus, VlsPLA(2) caused major alterations in the expression levels of genes encoding transcription factors and subtilisin-like proteases, which play a role in plant immunity. In conclusion, our study showed that VlsPLA(2) acts as a virulence factor, possibly by hydrolysing host nuclear envelope phospholipids, which, through a signal transduction cascade, may suppress basal plant immune responses

LysM Proteins Regulate Fungal Development and Contribute to Hyphal Protection and Biocontrol Traits in Clonostachys rosea

Lysin motif (LysM) modules are approximately 50 amino acids long and bind to peptidoglycan, chitin and its derivatives. Certain LysM proteins in plant pathogenic and entomopathogenic fungi are shown to scavenge chitin oligosaccharides and thereby dampen host defense reactions. Other LysM proteins can protect the fungal cell wall against hydrolytic enzymes. In this study, we investigated the biological function of LysM proteins in the mycoparasitic fungus Clonostachys rosea. The C. rosea genome contained three genes coding for LysM-containing proteins and gene expression analysis revealed that lysm1 and lysm2 were induced during mycoparasitic interaction with Fusarium graminearum and during colonization of wheat roots. Lysm1 was suppressed in germinating conidia, while lysm2 was induced during growth in chitin or peptidoglycan-containing medium. Deletion of lysm1 and lysm2 resulted in mutants with increased levels of conidiation and conidial germination, but reduced ability to control plant diseases caused by F. graminearum and Botrytis cinerea. The Delta lysm2 strain showed a distinct, accelerated mycelial disintegration phenotype accompanied by reduced biomass production and hyphal protection against hydrolytic enzymes including chitinases, suggesting a role of LYSM2 in hyphal protection against chitinases. The Delta lysm2 and Delta lysm1 Delta lysm2 strains displayed reduced ability to colonize wheat roots, while only Delta lysm1 Delta lysm2 failed to suppress expression of the wheat defense response genes PR1 and PR4. Based on our data, we propose a role of LYSM1 as a regulator of fungal development and of LYSM2 in cell wall protection against endogenous hydrolytic enzymes, while both are required to suppress plant defense responses. Our findings expand the understanding of the role of LysM proteins in fungal-fungal interactions and biocontrol

Transformation and gene-disruption in the apple-pathogen, Neonectria ditissima

Background Apple production in Sweden and elsewhere is being threatened by the fungus, Neonectria ditissima, which causes a disease known as European canker. The disease can cause extensive damage and the removal of diseased wood and heavily infected trees can be laborious and expensive. Currently, there is no way to eradicate the fungus from infected trees and our knowledge of the infection process is limited. Thus, to target and modify genes efficiently, the genetic transformation technique developed for N. ditissima back in 2003 was modified. Results The original protocol from 2003 was upgraded to use enzymes currently available in the market for making protoplasts. The protoplasts were viable, able to uptake foreign DNA, and able to regenerate back into a mycelial colony, either as targeted gene-disruption mutants or as ectopic mutants expressing the green fluorescent protein (GFP). Conclusions A new genetic transformation protocol has been established and the inclusion of hydroxyurea in the buffer during the protoplast-generation step greatly increased the creation of knockout mutants via homologous recombination. Pathogenicity assays using the GFP-mutants showed that the mutants were able to infect the host and cause disease

Insight into trade‐off between wood decay and parasitism from the genome of a fungal forest pathogen

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91191/1/j.1469-8137.2012.04128.x.pd

The Role of Glycoside Hydrolases in Phytopathogenic Fungi and Oomycetes Virulence

Phytopathogenic fungi need to secrete different hydrolytic enzymes to break down complex polysaccharides in the plant cell wall in order to enter the host and develop the disease. Fungi produce various types of cell wall degrading enzymes (CWDEs) during infection. Most of the characterized CWDEs belong to glycoside hydrolases (GHs). These enzymes hydrolyze glycosidic bonds and have been identified in many fungal species sequenced to date. Many studies have shown that CWDEs belong to several GH families and play significant roles in the invasion and pathogenicity of fungi and oomycetes during infection on the plant host, but their mode of function in virulence is not yet fully understood. Moreover, some of the CWDEs that belong to different GH families act as pathogen-associated molecular patterns (PAMPs), which trigger plant immune responses. In this review, we summarize the most important GHs that have been described in eukaryotic phytopathogens and are involved in the establishment of a successful infection

Anastomosis Is Required for Virulence of the Fungal Necrotroph Alternaria brassicicola▿

A fungal mycelium is typically composed of radially extending hyphal filaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination of nutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogen Alternaria brassicicola and two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of the A. brassicicola isolates tested, as well as A. mimicula and A. japonica, were capable of self-fusion, with two isolates of A. brassicicola being capable of non-self-fusion. Disruption of the anastomosis gene homolog (Aso1) in A. brassicicola resulted in both the loss of self-anastomosis and pathogenicity on cabbage. This finding, combined with our discovery that a previously described nonpathogenic A. brassicicola mutant defective for a mitogen-activated protein kinase gene (amk1) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity in A. brassicicola