fungal plant pathogens remain a serious threat to the sustainable agriculture and forestry, despite the extensive efforts undertaken to control their spread. White root rot disease is threatening rubber tree (Hevea brasiliensis) plantations throughout South and Southeast Asia and Western Africa, causing tree mortality and severe yield losses. Here, we report the complete genome sequence of the basidiomycete fungus Rigidoporus microporus, a causative agent of the disease. Our phylogenetic analysis confirmed the position of R. microporus among the members of Hymenochaetales, an understudied group of basidiomycetes. Our analysis further identified pathogen's genes with a predicted role in the decay of plant cell wall polymers, in the utilization of latex components and in interspecific interactions between the pathogen and other fungi. We also detected putative horizontal gene transfer events in the genome of R. microporus. The reported first genome sequence of a tropical rubber tree pathogen R. microporus should contribute to the better understanding of how the fungus is able to facilitate wood decay and nutrient cycling as well as tolerate latex and utilize resinous extractives.The white rot fungus known as Rigidoporus microporus (Sw.) Overeem (Basidiomycota, Agaricomycotina) is the most economically important pathogen of the tropical tree Hevea brasiliensis (Willd. ex A. Juss.) Müll.Arg., also known as Para rubber. Para rubber is principally valued for its latex content; the latex or natural rubber is a very significant industrial commodity used in a variety of industries from car manufacturing to healthcare. The market value of global annual production of natural rubber is over US$ 50 billion. Elastomers derived from natural rubber are indispensable in space, water, and ship technologies 1 . A major part of the plantation forestry in Africa and Asia is the growing of Para rubber.Rigidoporus microporus is therefore a fungal species of significant economic importance. As a necrotrophic pathogen, it has an extensive host range and affects many tropical and subtropical trees, food and cash crops 2,3 . However, it is best known as a causative agent of the white root rot disease of the rubber tree and for the devastating impact it has on commercial rubber tree plantations ( Fig. 1) 4 . The fungus produces rhizomorphs (thread-like mycelial aggregation of fungal hypha), which can grow several meters in the soil before they reach and attack roots of a suitable host, and it continues to rot wood long after the tree has fallen 5 . Population genetic studies suggested that a host jump and the lack of coevolution between the host and the pathogen might be the possible reasons for the aggressive behavior of the pathogen on rubber trees. This is further supported by the fact that the disease is devastating in Asia and West Africa, but not a serious problem in South America, the centre of origin of the rubber tree host 3 . Presence of high density of fungal rhizomorphs of R. microporus in the soil indicates a capacity to switch...

Heterobasidion parviporum is the most devastating fungal pathogen of conifer forests in Northern Europe. The fungus has dual life strategies, necrotrophy on living trees and saprotrophy on dead woods. DNA cytosine methylation is an important epigenetic modification in eukaryotic organisms. Our presumption is that the lifestyle transition and asexual development in H. parviporum could be driven by epigenetic effects. Involvements of DNA methylation in the regulation of aforementioned processes have never been studied thus far. RNA-seq identified lists of highly induced genes enriched in carbohydrate-active enzymes during necrotrophic interaction with host trees and saprotrophic sawdust growth. It also highlighted signaling-and transcription factor-related genes potentially associated with the transition of saprotrophic to necrotrophic lifestyle and groups of primary cellular activities throughout asexual development. Whole-genome bisulfite sequencing revealed that DNA methylation displayed pronounced preference in CpG dinucleotide context across the genome and mostly targeted transposable element (TE)-rich regions. TE methylation level demonstrated a strong negative correlation with TE expression, reinforcing the protective function of DNA methylation in fungal genome stability. Small groups of genes putatively subject to methylation transcriptional regulation in response to saprotrophic and necrotrophic growth in comparison with free-living mycelia were also explored. Our study reported on the first methylome map of a forest pathogen. Analysis of transcriptome and methylome variations associated with asexual development and different lifestyle strategies provided further understanding of basic biological processes in H. parviporum. More importantly, our work raised additional potential roles of DNA methylation in fungi apart from controlling the proliferation of TEs.

Heterobasidion parviporum Niemelä & Korhonen is a necrotrophic fungal pathogen of Norway spruce (Picea abies). H. parviporum genome encodes numerous necrotrophic small secreted proteins (SSP) which might be important for promoting and sustaining the disease development. However, their transcriptional dynamics and plant defense response during infection are largely unknown. In this study, we identified a necrotrophic SSP named HpSSP35.8 and its coding gene was highly expressed in the pre-symptomatic phase of host infection. We explored the impact of HpSSP35.8 on Nicotiana benthamiana using Agrobacterium-mediated transient expression system under visible spectrum RGB imaging and chlorophyll fluorescence imaging. The results showed that HpSSP35.8 triggered a form of SSP-associated programmed cell death, accompanied by a decrease in the plant photosynthetic activity. Defense-related genes including WRKY12, ethylene response factor (ERF1α) and a chitinase gene PR4 were up-regulated in both HpSSP35.8-N. benthamiana interaction and H. parviporum-Norway spruce pathosystem. It highlighted the potential for the use of chlorophyll fluorescence imaging approach for monitoring indirect effects of SSP as well as for selection of other potential effector-like protein candidates.

Forest trees frequently interact with a diverse range of microorganisms including dark septate root endophytes (DSE) and fungal pathogens. Plant defense response to either individual pathogen or endophyte has been widely studied, but very little is known on the effect of coinfection on host defenses. To study the impact of coinfection or tripartite interaction on plant growth and host defenses, Norway spruce (Picea abies) seedlings were inoculated with a DSE Phialocephala sphaeroides or with a root pathogen Heterobasidion parviporum, or coinfected with both fungi. The results showed that the root endophyte promoted the root growth of spruce seedlings. Control seedlings without any inoculum were subjected to sequencing and used as a baseline for identification of differentially expressed genes. RNA-Seq analysis of seedlings inoculated with P. sphaeroides, infected with H. parviporum or coinfected with both fungi resulted in a total of 5,269 differentially expressed genes (DEGs). The majority of DEGs were found in P. sphaeroides-inoculated seedlings. Lignin biosynthesis pathways were generally activated during fungal infections. The pattern was distinct with endophyte inoculation. The majority of the genes in the flavonoid biosynthesis pathway were generally suppressed during fungal infections. A specific transcriptional response to P. sphaeroides inoculation was the increased transcripts of genes involved in jasmonic acid biosynthesis, MAPK signaling pathway, plant hormone signal transduction, and calcium-mediated signaling. This may have potentially contributed to promoting the root growth of seedlings. Although the coinfection suppressed the induction of numerous genes, no negative effect on the growth of the spruce seedlings occurred. We conclude that the subsequent H. parviporum infection triggered reprogramming of host metabolism. Conversely, the endophyte (P. sphaeroides), on the other hand, counteracted the negative effects of H. parviporum on the growth of the spruce seedlings.

Heterobasidion parviporum Niemelä & Korhonen is an economically important basidiomycete, causing root and stem rot disease of Norway spruce (Picea abies (L.) Karst) in Northern Europe. The H. parviporum genome encodes numerous small secreted proteins, which might be of importance for interacting with mycorrhiza symbionts, endophytes, and other saprotrophs. We hypothesized that small secreted proteins from H. parviporum (HpSSPs) are involved in interspecific fungal interaction. To identify HpSSP-coding genes potentially involved, we screened the H. parviporum effectome and compared their transcriptomic profiles during fungal development and in planta tree infection. We further conducted phylogenetic analysis, and identified a subset of hypothetical proteins with nonpredicted domain or unknown function as HpSSPs candidates for further characterization. The HpSSPs candidates were selected based on high-quality sequence, cysteine residue frequency, protein size, and in planta expression. We subsequently explored their roles during in vitro interaction in paired cultures of H. parviporum with ectomycorrhizal Cortinarius gentilis, endophytic Phialocephala sphaeroides, saprotrophs (Mycena sp., Phlebiopsis gigantea, and Phanerochaete chrysosporium), respectively. The transcriptomic profile revealed that a large proportion of effector candidates was either barely expressed or highly expressed under all growth conditions. In vitro dual-culture test showed that P. sphaeroides and C. gentilis were overgrown by H. parviporum. The barrage zone formation or no physical contact observed in paired cultures with the saprotrophs suggest they had either combative interaction or antibiosis effect with H. parviporum. Several HpSSPs individuals were up- or downregulated during the nonself interactions. The results of HpSSPs gene expression patterns provide additional insights into the diverse roles of SSPs in tree infection and interspecific fungal interactions.