Small Secreted Proteins Research Articles

High rate of gene family evolution in proximity to the origin of ectomycorrhizal symbiosis in Inocybaceae.

The genomes of ectomycorrhizal (ECM) fungi have a reduced number of genes encoding Carbohydrate-Active EnZymes (CAZymes), expansions in transposable elements (TEs) and small secreted proteins (SSPs) compared with saprotrophs. Fewer genes for specific peptidases and lipases in ECM fungi are also reported. It is unclear whether these changes occur at the shift to the ECM habit or are more gradual throughout the evolution of ECM lineages. We generated a genomic dataset of 20 species in the ECM lineage Inocybaceae and compared them with six saprotrophic species. Inocybaceae genomes have fewer CAZymes, peptidases, lipases, secondary metabolite clusters and SSPs and higher TE content than their saprotrophic relatives. There was an increase in the rate of gene family evolution along the branch with the transition to the ECM lifestyle. This branch had very high rate of evolution in CAZymes and had the largest number of contractions. Other significant changes along this branch included expansions in transporters, transposons-related genes and communication genes such as fungal kinases. There is a high concentration of changes in proximity to the transition to the ECM lifestyle, which correspond to the identified key changes for the gain of this lifestyle.

The cysteine-rich virulence factor NipA of Arthrobotrys flagrans interferes with cuticle integrity of Caenorhabditis elegans

Animals protect themself from microbial attacks by robust skins or a cuticle as in Caenorhabditis elegans. Nematode-trapping fungi, like Arthrobotrys flagrans, overcome the cuticle barrier and colonize the nematode body. While lytic enzymes are important for infection, small-secreted proteins (SSPs) without enzymatic activity, emerge as crucial virulence factors. Here, we characterized NipA (nematode induced protein) which A. flagrans secretes at the penetration site. In the absence of NipA, A. flagrans required more time to penetrate C. elegans. Heterologous expression of the fungal protein in the epidermis of C. elegans led to blister formation. NipA contains 13 cysteines, 12 of which are likely to form disulfide bridges, and the remaining cysteine was crucial for blister formation. We hypothesize that NipA interferes with cuticle integrity to facilitate fungal entry. Genome-wide expression analyses of C. elegans expressing NipA revealed mis-regulation of genes associated with extracellular matrix (ECM) maintenance and innate immunity.

Chromosome-Level Assembly and Comparative Genomic Analysis of Suillus bovinus Provides Insights into the Mechanism of Mycorrhizal Symbiosis.

Suillus bovinus is a wild edible ectomycorrhizal fungus with important economic and ecological value, which often forms an ectomycorrhiza with pine trees. We know little about the mechanisms associated with the metabolism and symbiosis of S. bovinus and its effects on the nutritional value. In this study, the whole-genome sequencing of S. bovinus was performed using Illumina, HiFi, and Hi-C technologies, and the sequencing data were subjected to genome assembly, gene prediction, and functional annotation to obtain a high-quality chromosome-level genome of S. bovinus. The final assembly of the S. bovinus genome includes 12 chromosomes, with a total length of 43.03 Mb, a GC content of 46.58%, and a contig N50 size of 3.78 Mb. A total of 11,199 coding protein sequences were predicted from genome annotation. The S. bovinus genome contains a large number of small secreted proteins (SSPs) and genes that encode enzymes related to carbohydrates, as well as genes related to terpenoids, auxin, and lipochitooligosaccharides. These genes may contribute to symbiotic processes. The whole-genome sequencing and genetic information provide a theoretical basis for a deeper understanding of the mechanism of the mycorrhizal symbiosis of S. bovinus and can serve as a reference for comparative genomics of ectomycorrhizal fungi.

Altered Expression of Two Small Secreted Proteins (ssp4 and ssp6) Affects the Degradation of a Natural Lignocellulosic Substrate by Pleurotus ostreatus

Pleurotus ostreatus is a white-rot fungus that can degrade lignin in a preferential manner using a variety of extracellular enzymes, including manganese and versatile peroxidases (encoded by the vp1-3 and mnp1-6 genes, respectively). This fungus also secretes a family of structurally related small secreted proteins (SSPs) encoded by the ssp1-6 genes. Using RNA sequencing (RNA-seq), we determined that ssp4 and ssp6 are the predominant members of this gene family that were expressed by P. ostreatus during the first three weeks of growth on wheat straw. Downregulation of ssp4 in a strain harboring an ssp RNAi construct (KDssp1) was then confirmed, which, along with an increase in ssp6 transcript levels, coincided with reduced lignin degradation and the downregulation of vp2 and mnp1. In contrast, we observed an increase in the expression of genes related to pectin and side-chain hemicellulose degradation, which was accompanied by an increase in extracellular pectin-degrading capacity. Genome-wide comparisons between the KDssp1 and the wild-type strains demonstrated that ssp silencing conferred accumulated changes in gene expression at the advanced cultivation stages in an adaptive rather than an inductive mode of transcriptional response. Based on co-expression networking, crucial gene modules were identified and linked to the ssp knockdown genotype at different cultivation times. Based on these data, as well as previous studies, we propose that P. ostreatus SSPs have potential roles in modulating the lignocellulolytic and pectinolytic systems, as well as a variety of fundamental biological processes related to fungal growth and development.

AlphaFold-Multimer predicts cross-kingdom interactions at the plant-pathogen interface

Adapted plant pathogens from various microbial kingdoms produce hundreds of unrelated small secreted proteins (SSPs) with elusive roles. Here, we used AlphaFold-Multimer (AFM) to screen 1879 SSPs of seven tomato pathogens for interacting with six defence-related hydrolases of tomato. This screen of 11,274 protein pairs identified 15 non-annotated SSPs that are predicted to obstruct the active site of chitinases and proteases with an intrinsic fold. Four SSPs were experimentally verified to be inhibitors of pathogenesis-related subtilase P69B, including extracellular protein-36 (Ecp36) and secreted-into-xylem-15 (Six15) of the fungal pathogens Cladosporium fulvum and Fusarium oxysporum, respectively. Together with a P69B inhibitor from the bacterial pathogen Xanthomonas perforans and Kazal-like inhibitors of the oomycete pathogen Phytophthora infestans, P69B emerges as an effector hub targeted by different microbial kingdoms, consistent with a diversification of P69B orthologs and paralogs. This study demonstrates the power of artificial intelligence to predict cross-kingdom interactions at the plant-pathogen interface.

The neighbouring genes AvrLm10A and AvrLm10B are part of a large multigene family of cooperating effector genes conserved in Dothideomycetes and Sordariomycetes.

Fungal effectors (small-secreted proteins) have long been considered as species or even subpopulation-specific. The increasing availability of high-quality fungal genomes and annotations has allowed the identification of trans-species or trans-genera families of effectors. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus causing stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighbouring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome showed that AvrLm10A/AvrLm10B belong to a multigene family comprising five pairs of genes with a similar tail-to-tail organization. The two genes, in a pair, always had the same expression pattern and two expression profiles were distinguished, associated with the biotrophic colonization of cotyledons and/or petioles and stems. Of the two protein pairs further investigated, AvrLm10A_like1/AvrLm10B_like1 and AvrLm10A_like2/AvrLm10B_like2, the second one had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair, and cross-interactions were also detected for two pairs. AvrLm10A homologues were identified in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi. One of them, SIX5, is an effector from Fusarium oxysporum f. sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A/SIX5 homologues were associated with at least eight distinct putative effector families, suggesting that AvrLm10A/SIX5 is able to cooperate with different effectors. These results point to a general role of the AvrLm10A/SIX5 proteins as "cooperating proteins", able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighbouring AvrLm10A homologue.

Physiological function of hydrophobin Vmh3 in lignin degradation by white-rot fungus Pleurotus ostreatus.

Hydrophobins are small-secreted proteins comprising both hydrophobic and hydrophilic parts, that can self-assemble into an amphiphilic film at the air-liquid interface. More than 20 hydrophobin genes have been estimated in the white-rot fungus Pleurotus ostreatus. In our previous studies, three hydrophobin genes were shown to be predominantly expressed under ligninolytic conditions, and only vmh3 was downregulated in both the delignification-deficient mutant Δgat1 and Δhir1 strains. Here, we focused on the function of the hydrophobin Vmh3 to clarify its physiological role in lignin degradation. When the hyphae were observed by transmission electron microscopy, deletion of vmh3 resulted in the disappearance of black aggregates at the interface between the cell wall and outer environment. Deletion of vmh3 resulted in reduced hydrophobicity when 0.2% sodium dodecyl sulfate was dropped onto the mycelial surface. These results suggest that Vmh3 functions on the cell surface and plays a major role in mycelial hydrophobization. Furthermore, the Δvmh3 strain showed a marked delay in lignin degradation on beech wood sawdust medium, while the production of lignin-modifying enzymes was not reduced. This study demonstrated, for the first time, the possible effect of hydrophobin on lignin degradation by a white-rot fungus.

Speciation Underpinned by Unexpected Molecular Diversity in the Mycorrhizal Fungal Genus Pisolithus.

The mutualistic ectomycorrhizal (ECM) fungal genus Pisolithus comprises 19 species defined to date which colonize the roots of >50 hosts worldwide suggesting that substantial genomic and functional evolution occurred during speciation. To better understand this intra-genus variation, we undertook a comparative multi-omic study of nine Pisolithus species sampled from North America, South America, Asia, and Australasia. We found that there was a small core set of genes common to all species (13%), and that these genes were more likely to be significantly regulated during symbiosis with a host than accessory or species-specific genes. Thus, the genetic "toolbox" foundational to the symbiotic lifestyle in this genus is small. Transposable elements were located significantly closer to gene classes including effector-like small secreted proteins (SSPs). Poorly conserved SSPs were more likely to be induced by symbiosis, suggesting that they may be a class of protein that tune host specificity. The Pisolithus gene repertoire is characterized by divergent CAZyme profiles when compared with other fungi, both symbiotic and saprotrophic. This was driven by differences in enzymes associated with symbiotic sugar processing, although metabolomic analysis suggest that neither copy number nor expression of these genes is sufficient to predict sugar capture from a host plant or its metabolism in fungal hyphae. Our results demonstrate that intra-genus genomic and functional diversity within ECM fungi is greater than previously thought, underlining the importance of continued comparative studies within the fungal tree of life to refine our focus on pathways and evolutionary processes foundational to this symbiotic lifestyle.

Comparative Transcriptomics Profiling of Perennial Ryegrass Infected with Wild Type or a ΔvelA Epichloë festucae Mutant Reveals Host Processes Underlying Mutualistic versus Antagonistic Interactions

Epichloë species form bioprotective endophytic symbioses with many cool-season grasses, including agriculturally important forage grasses. Despite its importance, relatively little is known about the molecular details of the interaction and the regulatory genes involved. VelA is a key global regulator in fungal secondary metabolism and development. In previous studies, we showed the requirement of velA for E. festucae to form a mutualistic interaction with Lolium perenne. We showed that VelA regulates the expression of genes encoding proteins involved in membrane transport, fungal cell wall biosynthesis, host cell wall degradation, and secondary metabolism, along with several small-secreted proteins in Epichloë festucae. Here, by a comparative transcriptomics analysis on perennial ryegrass seedlings and mature plants, which are endophyte free or infected with wild type (mutualistic interaction) or mutant ΔvelA E. festucae (antagonistic or incompatible interaction), regulatory effects of the endophytic interaction on perennial ryegrass development was studied. We show that ΔvelA mutant associations influence the expression of genes involved in primary metabolism, secondary metabolism, and response to biotic and abiotic stresses compared with wild type associations, providing an insight into processes defining mutualistic versus antagonistic interactions.

Features of disruption mutants of genes encoding for hydrophobin Vmh2 and Vmh3 in mycelial formation and resistance to environmental stress in Pleurotus ostreatus.

Hydrophobins, which are small-secreted proteins with both hydrophobic and hydrophilic parts, can self-assemble into an amphiphilic film at the air-water interface, helping the fungus to form aerial hyphae. In the agaricomycete Pleurotus ostreatus, more than 20 putative hydrophobin genes have been predicted. Of these, two hydrophobin genes, vmh2 and vmh3, are predominantly expressed in the vegetative mycelium. In this study, we focused on the functions of Vmh2 and Vmh3 in vegetative mycelia. Based on the observation of the mycelial cross-section by transmission electron microscopy and the disappearance time of water droplets on the mycelial surface, Vmh2 and Vmh3 were considered essential for the maintenance of the surface hydrophobicity of the mycelium. The Δvmh3 and Δvmh2Δvmh3 strains exhibited relatively slower aerial mycelia formation on a liquid medium, and no significant alteration was observed in Δvmh2 strains. Only the Δvmh3 and Δvmh2Δvmh3 strains grew slower than the wild-type strain under stress conditions involving SDS and H2O2 on agar plates. This study revealed possible distinct roles for these hydrophobins in stress resistance. These results suggest that Agaricomycetes, including P. ostreatus, have evolved to possess multiple different hydrophobins as a means of adapting to various environments.

Secretomic Insights into the Pathophysiology of Venturia inaequalis: The Causative Agent of Scab, a Devastating Apple Tree Disease

Apple scab, caused by Venturia inaequalis, is one of the world's most commercially significant apple diseases. The fungi have a catastrophic impact on apples, causing considerable losses in fruit quality and productivity in many apple-growing locations despite numerous control agents. Fungi secrete various effectors and other virulence-associated proteins that suppress or alter the host's immune system, and several such proteins were discovered in this work. Using state-of-the-art bioinformatics techniques, we examined the V. inaequalis reference genome (EU-B04), resulting in the identification of 647 secreted proteins, of which 328 were classified as small secreted proteins (SSPs), with 76.52% of SSPs identified as anticipated effector proteins. The more prevalent CAZyme proteins were the enzymes engaged in plant cell wall disintegration (targeting pectin and xylanase), adhesion and penetration (Cutinases/acetyl xylan esterase), and reactive oxygen species formation (multicopper oxidases). Furthermore, members of the S9 prolyl oligopeptidase family were identified as the most abundant host defense peptidases. Several known effector proteins were discovered to be expressed during the V. inaequalis infection process on apple leaves. The present study provides valuable data that can be used to develop new strategies for controlling apple scab.

Identification of Gene Modules and Hub Genes Associated with Sporisorium scitamineum Infection Using Weighted Gene Co-Expression Network Analysis.

Sporisorium scitamineum is a biotrophic fungus responsible for sugarcane smut disease. To investigate the key genes involved in S. scitamineum infection, we conducted RNA sequencing of sugarcane sprouts inoculated with S. scitamineum teliospores. A weighted gene co-expression network analysis (WGCNA) showed that two co-expressed gene modules, MEdarkturquoise and MEpurple—containing 66 and 208 genes, respectively—were associated with S. scitamineum infection. The genes in these two modules were further studied using Gene Ontology (GO) enrichment analysis, pathogen-host interaction (PHI) database BLASTp, and small secreted cysteine-rich proteins (SCRPs) prediction. The top ten hub genes in each module were identified using the Cytohubba plugin. The GO enrichment analysis found that endoplasmic reticulum-related and catabolism-related genes were expressed during S. scitamineum infection. A total of 83 genes had homologs in the PHI database, 62 of which correlated with pathogen virulence. A total of 21 proteins had the characteristics of small secreted cysteine-rich proteins (SCRPs), a common source of fungal effectors. The top ten hub genes in each module were identified, and seven were annotated as Mig1-Mig1 protein, glycosyl hydrolase, beta-N-acetylglucosaminidase, secreted chorismate mutase, collagen, mRNA export factor, and pleckstrin homology domain protein, while the remaining three were unknown. Two SCRPs—SPSC_06609 and SPSC_04676—and three proteins—SPSC_01958, SPSC_02155, and SPSC_00940—identified in the PHI database were also among the top ten hub genes in the MEdarkturquoise and MEpurple modules, suggesting that they may play important roles in S. scitamineum infection. A S. scitamineum infection model was postulated based on current findings. These findings help to deepen the current understanding of early events in S. scitamineum infection.

Aspergillus Hydrophobins: Physicochemical Properties, Biochemical Properties, and Functions in Solid Polymer Degradation.

Hydrophobins are small amphipathic proteins conserved in filamentous fungi. In this review, the properties and functions of Aspergillus hydrophobins are comprehensively discussed on the basis of recent findings. Multiple Aspergillus hydrophobins have been identified and categorized in conventional class I and two non-conventional classes. Some Aspergillus hydrophobins can be purified in a water phase without organic solvents. Class I hydrophobins of Aspergilli self-assemble to form amphipathic membranes. At the air–liquid interface, RolA of Aspergillus oryzae self-assembles via four stages, and its self-assembled films consist of two layers, a rodlet membrane facing air and rod-like structures facing liquid. The self-assembly depends mainly on hydrophobin conformation and solution pH. Cys4–Cys5 and Cys7–Cys8 loops, disulfide bonds, and conserved Cys residues of RodA-like hydrophobins are necessary for self-assembly at the interface and for adsorption to solid surfaces. AfRodA helps Aspergillus fumigatus to evade recognition by the host immune system. RodA-like hydrophobins recruit cutinases to promote the hydrolysis of aliphatic polyesters. This mechanism appears to be conserved in Aspergillus and other filamentous fungi, and may be beneficial for their growth. Aspergilli produce various small secreted proteins (SSPs) including hydrophobins, hydrophobic surface–binding proteins, and effector proteins. Aspergilli may use a wide variety of SSPs to decompose solid polymers.

Small-secreted proteins as virulence factors in nematode-trapping fungi.

Nematode-trapping fungi (NTF), such as Arthrobotrys flagrans (Duddingtonia flagrans), are soil-borne fungi able to form adhesive trapping networks to attract and catch nematodes. In this forum piece we highlight some of their most fascinating features with a special focus on the role of small-secreted proteins in the predatory interaction.

Comparative Genomics of Three Aspergillus Strains Reveals Insights into Endophytic Lifestyle and Endophyte-Induced Plant Growth Promotion.

Aspergillus includes both plant pathogenic and beneficial fungi. Although endophytes beneficial to plants have high potential for plant growth promotion and improving stress tolerance, studies on endophytic lifestyles and endophyte-plant interactions are still limited. Here, three endophytes belonging to Aspergillus, AS31, AS33, and AS42, were isolated. They could successfully colonize rice roots and significantly improved rice growth. The genomes of strains AS31, AS33, and AS42 were sequenced and compared with other Aspergillus species covering both pathogens and endophytes. The genomes of AS31, AS33, and AS42 were 36.8, 34.8, and 35.3 Mb, respectively. The endophytic genomes had more genes encoding carbohydrate-active enzymes (CAZymes) and small secreted proteins (SSPs) and secondary metabolism gene clusters involved in indole metabolism than the pathogens. In addition, these endophytes were able to improve Pi (phosphorus) accumulation and transport in rice by inducing the expression of Pi transport genes in rice. Specifically, inoculation with endophytes significantly increased Pi contents in roots at the early stage, while the Pi contents in inoculated shoots were significantly increased at the late stage. Our results not only provide important insights into endophyte-plant interactions but also provide strain and genome resources, paving the way for the agricultural application of Aspergillus endophytes.

MAPK Cascades Mediating Biocontrol Activity of Trichoderma brevicrassum Strain TC967.

Trichoderma brevicrassum strain TC967 is a novel biological control agent (BCA) against the plant pathogen Rhizoctonia solani and promotes plant growth. Mitogen-activated protein kinase (MAPK) cascades are involved in a variety of physiological functions of Trichoderma, but functions of each MAPK in regulating biocontrol have not been characterized in a single Trichoderma strain. In this study, we assembled and annotated the genome of strain TC967 and identified its three MAPK gene sequences. Functions of Fus3-, Slt2-, and Hog1-MAPK in strain TC967 were dissected. All three MAPKs were involved in hyphal growth. Hog1-MAPK was essential for conidiation and tolerance to saline/osmotic stress. Both Fus3- and Slt2-MAPK deletion mutants reduced cell-wall integrity (CWI) and increased the activities of chitinase and protease. The growth of Rhizoctoniasolani was further inhibited by volatile organic compounds (VOCs) and secondary metabolites produced by Fus3- and Slt2-MAPK deletion mutants, respectively. Biocontrol assays demonstrated that Fus3- and Slt2-MAPK deletion mutants were considerably more effective in disease control than the wild-type strain. RNA-seq analysis revealed that MAPK collectively played a major role in regulating biocontrol-related gene expressions, including the genes in charge of secondary metabolism, fungal cell wall-degrading enzymes (FCWDEs), and small secreted cysteine-rich proteins (SSCPs).

A Comprehensive Assessment of the Secretome Responsible for Host Adaptation of the Legume Root Pathogen Aphanomyces euteiches.

The soil-borne oomycete pathogen Aphanomyces euteiches causes devastating root rot diseases in legumes such as pea and alfalfa. The different pathotypes of A. euteiches have been shown to exhibit differential quantitative virulence, but the molecular basis of host adaptation has not yet been clarified. Here, we re-sequenced a pea field reference strain of A. euteiches ATCC201684 with PacBio long-reads and took advantage of the technology to generate the mitochondrial genome. We identified that the secretome of A. euteiches is characterized by a large portfolio of secreted proteases and carbohydrate-active enzymes (CAZymes). We performed Illumina sequencing of four strains of A. euteiches with contrasted specificity to pea or alfalfa and found in different geographical areas. Comparative analysis showed that the core secretome is largely represented by CAZymes and proteases. The specific secretome is mainly composed of a large set of small, secreted proteins (SSP) without any predicted functional domain, suggesting that the legume preference of the pathogen is probably associated with unknown functions. This study forms the basis for further investigations into the mechanisms of interaction of A. euteiches with legumes.

Evolutionary transition to the ectomycorrhizal habit in the genomes of a hyperdiverse lineage of mushroom-forming fungi.

The ectomycorrhizal (ECM) symbiosis has independently evolved from diverse types of saprotrophic ancestors. In this study, we seek to identify genomic signatures of the transition to the ECM habit within the hyperdiverse Russulaceae. We present comparative analyses of the genomic architecture and the total and secreted gene repertoires of 18 species across the order Russulales, of which 13 are newly sequenced, including a representative of a saprotrophic member of Russulaceae, Gloeopeniophorella convolvens. The genomes of ECM Russulaceae are characterized by a loss of genes for plant cell wall-degrading enzymes (PCWDEs), an expansion of genome size through increased transposable element (TE) content, a reduction in secondary metabolism clusters, and an association of small secreted proteins (SSPs) with TE 'nests', or dense aggregations of TEs. Some PCWDEs have been retained or even expanded, mostly in a species-specific manner. The genome of G. convolvens possesses some characteristics of ECM genomes (e.g. loss of some PCWDEs, TE expansion, reduction in secondary metabolism clusters). Functional specialization in ECM decomposition may drive diversification. Accelerated gene evolution predates the evolution of the ECM habit, indicating that changes in genome architecture and gene content may be necessary to prime the evolutionary switch.

Diversity and conservation of plant small secreted proteins associated with arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal symbiosis (AMS) is widespread mutualistic association between plants and fungi, which plays an essential role in nutrient exchange, enhancement in plant stress resistance, development of host, and ecosystem sustainability. Previous studies have shown that plant small secreted proteins (SSPs) are involved in beneficial symbiotic interactions. However, the role of SSPs in the evolution of AMS has not been well studied yet. In this study, we performed computational analysis of SSPs in 60 plant species and identified three AMS-specific ortholog groups containing SSPs only from at least 30% of the AMS species in this study and three AMS-preferential ortholog groups containing SSPs from both AMS and non-AMS species, with AMS species containing significantly more SSPs than non-AMS species. We found that independent lineages of monocot and eudicot plants contained genes in the AMS-specific ortholog groups and had significant expansion in the AMS-preferential ortholog groups. Also, two AMS-preferential ortholog groups showed convergent changes, between monocot and eudicot species, in gene expression in response to arbuscular mycorrhizal fungus Rhizophagus irregularis. Furthermore, conserved cis-elements were identified in the promoter regions of the genes showing convergent gene expression. We found that the SSPs, and their closely related homologs, in each of three AMS-preferential ortholog groups, had some local variations in the protein structural alignment. We also identified genes co-expressed with the Populus trichocarpa SSP genes in the AMS-preferential ortholog groups. This first plant kingdom-wide analysis on SSP provides insights on plant-AMS convergent evolution with specific SSP gene expression and local diversification of protein structures.

Whole genome sequencing of an edible and medicinal mushroom, Russula griseocarnosa, and its association with mycorrhizal characteristics.

Russula griseocarnosa is a well-known ectomycorrhizal mushroom, which is mainly distributed in the Southern China. Although several scholars have attempted to isolate and cultivate fungal strains, no accurate method for culture of artificial fruiting bodies has been presented owing to difficulties associated with mycelium growth on artificial media. Herein, we sequenced R. griseocarnosa genome using the second- and third-generation sequencing technologies, followed by de novo assembly of high-throughput sequencing reads, and GeneMark-ES, BLAST, CAZy, and other databases were utilized for functional gene annotation. We also constructed a phylogenetic tree using different species of fungi, and also conducted comparative genomics analysis of R. griseocarnosa against its four representative species. In addition, we evaluated the accuracy of one already sequenced genome of R. griseocarnosa based on the internal transcribed spacer (ITS) sequencing of that type of species. The assembly process resulted in identification of 230 scaffolds with a total genome size of 50.67 Mbp. The gene prediction showed that R. griseocarnosa genome included 14,229 coding sequences (CDs). In addition, 470 RNAs were predicted with 155 transfer RNAs (tRNAs), 49 ribosomal RNAs (rRNAs), 41 small noncoding RNAs (sRNAs), 42 small nuclear RNAs (snRNAs), and 183 microRNAs (miRNAs). The predicted protein sequences of R. griseocarnosa were analyzed to indicate the existence of carbohydrate-active enzymes (CAZymes), and the results revealed that 153 genes encoded CAZymes, which were distributed in 58 CAZyme families. These enzymes included 78 glycoside hydrolases (GHs), 34 glycosyl transferases (GTs), 30 auxiliary activities (AAs), 2 carbohydrate esterases (CEs), 8 carbohydrate-binding modules (CBMs), and only one polysaccharide lyase (PL). Compared with other fungi, R. griseocarnosa had fewer CAZymes, and the number and distribution of CAZymes were similar to other mycorrhizal fungi, such as Tricholoma matsutake and Suillus luteus. Well-defined effector proteins that were associated with mycorrhiza-induced small-secreted proteins (MiSSPs) were not found in R. griseocarnosa, which indicated that there may be some special effector proteins to interact with host plants in R. griseocarnosa. The genome of R. griseocarnosa may provide new insights into the energy metabolism of ectomycorrhizal (ECM) fungi, a reference to study ecosystem and evolutionary diversification of R. griseocarnosa, as well as promoting the study of artificial domestication.