Small Cysteine-rich Proteins Research Articles

Complete genomes of Asgard archaea reveal diverse integrated and mobile genetic elements.

Asgard archaea are of great interest as the progenitors of Eukaryotes, but little is known about the mobile genetic elements (MGEs) that may shape their ongoing evolution. Here, we describe MGEs that replicate in Atabeyarchaeia, a wetland Asgard archaea lineage represented by two complete genomes. We used soil depth-resolved population metagenomic data sets to track 18 MGEs for which genome structures were defined and precise chromosome integration sites could be identified for confident host linkage. Additionally, we identified a complete 20.67 kbp circular plasmid and two family-level groups of viruses linked to Atabeyarchaeia, via CRISPR spacer targeting. Closely related 40 kbp viruses possess a hypervariable genomic region encoding combinations of specific genes for small cysteine-rich proteins structurally similar to restriction-homing endonucleases. One 10.9 kbp integrative conjugative element (ICE) integrates genomically into the Atabeyarchaeum deiterrae-1 chromosome and has a 2.5 kbp circularizable element integrated within it. The 10.9 kbp ICE encodes an expressed Type IIG restriction-modification system with a sequence specificity matching an active methylation motif identified by Pacific Biosciences (PacBio) high-accuracy long-read (HiFi) metagenomic sequencing. Restriction-modification of Atabeyarchaeia differs from that of another coexisting Asgard archaea, Freyarchaeia, which has few identified MGEs but possesses diverse defense mechanisms, including DISARM and Hachiman, not found in Atabeyarchaeia. Overall, defense systems and methylation mechanisms of Asgard archaea likely modulate their interactions with MGEs, and integration/excision and copy number variation of MGEs in turn enable host genetic versatility.

Deleting an xylosidase-encoding gene VdxyL3 increases growth and pathogenicity of Verticillium dahlia.

Verticillium dahliae causes a devastating Verticillium wilt disease on hundreds of plant species worldwide, including cotton. Understanding the interaction mechanism between V. dahliae and its hosts is the prerequisite for developing effective strategies for disease prevention. Here, based on the previous observation of an xylosidase-encoding gene (VdxyL3) in V. dahliae being obviously up-regulated after sensing root exudates from a cotton variety susceptible to this pathogen, we investigated the function of VdxyL3 in the growth and pathogenesis of V. dahliae by generating its deletion-mutant strains (ΔVdxyL3). Deleting VdxyL3 led to increased colony expansion rate, conidial production, mycelial growth, carbon and nitrogen utilization capacities, and enhanced stress tolerance and pathogenicity of V. dahliae. VdxyL3 is a secretory protein; however, VdxyL3 failed to induce cell death in N. benthamiana based on transient expression experiment. Transcriptomic analysis identified 1300 genes differentially expressed (DEGs) between wild-type (Vd952) and ΔVdxyL3 during infection, including 348 DEGs encoding secretory proteins, among which contained 122 classical secreted proteins and 226 non-classical secreted proteins. It was notable that of the 122 classical secretory proteins, 50 were carbohydrate-active enzymes (CAZymes) and 58 were small cysteine rich proteins (SCRPs), which were required for the pathogenicity of V. dahliae. The RNA-seq data thus potentially connected the genes encoding these proteins to the pathogenesis of V. dahliae. This study provides an experimental basis for further studies on the interaction between V. dahliae and cotton and the pathogenic mechanism of the fungus.

Insights into Phakopsora pachyrhizi Effector-Effector Interactions.

The multifaceted role of pathogen-encoded effectors in plant-pathogen interactions is complex and not fully understood. Effectors operate within intricate host environments, interacting with host proteins and other effectors to modulate virulence. The complex interplay between effectors raises the concept of metaeffectors, wherein some effectors regulate the activity of others. While previous research has demonstrated the importance of effector repertoires in pathogen virulence, only a limited number of studies have investigated the interactions between these effectors. This study explores the interactions among Phakopsora pachyrhizi effector candidates (PpECs). P.pachyrhizi haustorial transcriptome analysis identified a collection of predicted PpECs. Among these, PpEC23 was found to interact with PpEC48, prompting further exploration into their potential interaction with other effectors. Here, we utilized a yeast two-hybrid screen to explore protein-protein interactions between PpECs. A split-luciferase complementation assay also demonstrated that these interactions could occur within soybean cells. Interestingly, PpEC48 displayed the ability to interact with several small cysteine-rich proteins (SCRPs), suggesting its affinity for this specific class of effectors. We show that these interactions involve a histidine-rich domain within PpEC48, emphasizing the significance of structural motifs in mediating effector interactions. The unique nature of PpEC48, showing no sequence matches in other organisms, suggests its relatively recent evolution and potential orphan gene status. Our work reveals insights into the intricate network of interactions among P. pachyrhizi effector-effector interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Evolutionary Analysis of Cnidaria Small Cysteine-Rich Proteins (SCRiPs), an Enigmatic Neurotoxin Family from Stony Corals and Sea Anemones (Anthozoa: Hexacorallia).

Cnidarians (corals, sea anemones, and jellyfish) produce toxins that play central roles in key ecological processes, including predation, defense, and competition, being the oldest extant venomous animal lineage. Cnidaria small cysteine-rich proteins (SCRiPs) were the first family of neurotoxins detected in stony corals, one of the ocean's most crucial foundation species. Yet, their molecular evolution remains poorly understood. Moreover, the lack of a clear classification system has hindered the establishment of an accurate and phylogenetically informed nomenclature. In this study, we extensively surveyed 117 genomes and 103 transcriptomes of cnidarians to identify orthologous SCRiP gene sequences. We annotated a total of 168 novel putative SCRiPs from over 36 species of stony corals and 12 species of sea anemones. Phylogenetic reconstruction identified four distinct SCRiP subfamilies, according to strict discrimination criteria based on well-supported monophyly with a high percentage of nucleotide and amino acids' identity. Although there is a high prevalence of purifying selection for most SCRiP subfamilies, with few positively selected sites detected, a subset of Acroporidae sequences is influenced by diversifying positive selection, suggesting potential neofunctionalizations related to the fine-tuning of toxin potency. We propose a new nomenclature classification system relying on the phylogenetic distribution and evolution of SCRiPs across Anthozoa, which will further assist future proteomic and functional research efforts.

Evidence of Small Fungal Cysteine-Rich Proteins Acting as Biosurfactants and Self-Assembling into Large Fibers.

Fungi produce surface-active proteins, among which hydrophobins are the most characterized and attractive also for their ability to form functional amyloids. Our most recent findings show that these abilities are shared with other classes of fungal proteins. Indeed, in this paper, we compared the characteristics of a class I hydrophobin (Vmh2 from Pleurotus ostreatus) and an unknown protein (named PAC3), extracted from the marine fungal strain Acremonium sclerotigenum, which does not belong to the same protein family based on its sequence features. They both proved to be good biosurfactants, stabilizing emulsions in several conditions (concentration, pH, and salinity) and decreasing surface tension to a comparable value to that of some synthetic surfactants. After that, we observed for both Vmh2 and PAC3 the formation of giant fibers without the need for harsh conditions or long incubation time, a remarkable ability herein reported for the first time.

The Verticillium dahliae Small Cysteine-Rich Protein VdSCP23 Manipulates Host Immunity.

Verticillium wilt caused by Verticillium dahliae is a notorious soil-borne fungal disease and seriously threatens the yield of economic crops worldwide. During host infection, V. dahliae secretes many effectors that manipulate host immunity, among which small cysteine-rich proteins (SCPs) play an important role. However, the exact roles of many SCPs from V. dahliae are unknown and varied. In this study, we show that the small cysteine-rich protein VdSCP23 inhibits cell necrosis in Nicotiana benthamiana leaves, as well as the reactive oxygen species (ROS) burst, electrolyte leakage and the expression of defense-related genes. VdSCP23 is mainly localized in the plant cell plasma membrane and nucleus, but its inhibition of immune responses was independent of its nuclear localization. Site-directed mutagenesis and peptide truncation showed that the inhibition function of VdSCP23 was independent of cysteine residues but was dependent on the N-glycosylation sites and the integrity of VdSCP23 protein structure. Deletion of VdSCP23 did not affect the growth and development of mycelia or conidial production in V. dahliae. Unexpectedly, VdSCP23 deletion strains still maintained their virulence for N. benthamiana, Gossypium hirsutum and Arabidopsis thaliana seedlings. This study demonstrates an important role for VdSCP23 in the inhibition of plant immune responses; however, it is not required for normal growth or virulence in V. dahliae.

From methodological limitations to the function of metallothioneins - A guide to approaches for determining weak, moderate, and tight affinity zinc sites.

Mammalian metallothioneins (MTs) are small cysteine-rich proteins whose primary role is participation in zinc and copper homeostasis. Ever since their discovery, MTs have been investigated in terms of metal-binding affinity. The initial concept of seven Zn(II) ions (Zn7MT) bound with the same, undifferentiated low picomolar affinity in the α and β domains prevailed for many years and derived from spectroscopic studies. The application of fluorescent zinc probes has changed the perception of MTs, showing that they function in nano- to subnanomolar free zinc concentrations due to the presence of tight, moderate, and weak binding sites. The discovery of Zn(II)-depleted MTs in many tissues and determination of cellular free Zn(II) concentrations with differentiated zinc affinity sites revealed the critical importance of partially saturated Zn4-6MTs species in cellular zinc buffering in a wide pico- to nanomolar range of free Zn(II) concentrations. Until today, there is no clear agreement on the presence of differentiated or only tight zinc sites. Here, we present a series of spectroscopic, mass spectrometry-based, and enzymatic competition experiments which reveal how weak, moderate, or high affinity ligands interact with human MT2, with special attention to the determination of Zn(II) affinities. The results show that the simplification of the stability model is the major reason for determining significantly different stability data that obscured the actual MTs function. Therefore, we emphasize that different metal affinities are the single most reason for their presumed function, which changed over the years from tight binding and, thus, storage to one that is highly dynamic.

The YBR056W-A and Its Ortholog YDR034W-B of S. cerevisiae Belonging to CYSTM Family Participate in Manganese Stress Overcoming

The CYSTM (cysteine-rich transmembrane module) protein family comprises small molecular cysteine-rich tail-anchored membrane proteins found in many eukaryotes. The Saccharomyces cerevisiae strains carrying the CYSTM genes YDRO34W-B and YBR056W-A (MNC1) fused with GFP were used to test the expression of these genes under different stresses. The YBR056W-A (MNC1) and YDR034W-B genes are expressed under stress conditions caused by the toxic concentrations of heavy metal ions, such as manganese, cobalt, nickel, zinc, cuprum, and 2.4-dinitrophenol uncoupler. The expression level of YDR034W-B was higher than that of YBR056W-A under alkali and cadmium stresses. The Ydr034w-b-GFP and Ybr056w-a-GFP proteins differ in the cellular localization: Ydr034w-b-GFP was mainly observed in the plasma membrane and vacuolar membrane, while Ybr056w-a-GFP was observed in the cytoplasm, probably in intracellular membranes. The null-mutants in both genes demonstrated decreased cell concentration and lytic phenotype when cultivated in the presence of excess manganese. This allows for speculations about the involvement of Mnc1 and Ydr034w-b proteins in manganese stress overcoming.

Time-Course Transcriptome Profiling Reveals Differential Resistance Responses of Tomato to a Phytotoxic Effector of the Pathogenic Oomycete Phytophthora cactorum

Blight caused by Phytophthora pathogens has a devastating impact on crop production. Phytophthora species secrete an array of effectors, such as Phytophthora cactorum-Fragaria (PcF)/small cysteine-rich (SCR) phytotoxic proteins, to facilitate their infections. Understanding host responses to such proteins is essential to developing next-generation crop resistance. Our previous work identified a small, 8.1 kDa protein, SCR96, as an important virulence factor in Phytophthora cactorum. Host responses to SCR96 remain obscure. Here, we analyzed the effect of SCR96 on the resistance of tomato treated with this recombinant protein purified from yeast cells. A temporal transcriptome analysis of tomato leaves infiltrated with 500 nM SCR96 for 0, 3, 6, and 12 h was performed using RNA-Seq. In total, 36,779 genes, including 2704 novel ones, were detected, of which 32,640 (88.7%) were annotated. As a whole, 5929 non-redundant genes were found to be significantly co-upregulated in SCR96-treated leaves (3, 6, 12 h) compared to the control (0 h). The combination of annotation, enrichment, and clustering analyses showed significant changes in expression beginning at 3 h after treatment in genes associated with defense and metabolism pathways, as well as temporal transcriptional accumulation patterns. Noticeably, the expression levels of resistance-related genes encoding receptor-like kinases/proteins, resistance proteins, mitogen-activated protein kinases (MAPKs), transcription factors, pathogenesis-related proteins, and transport proteins were significantly affected by SCR96. Quantitative reverse transcription PCR (qRT-PCR) validated the transcript changes in the 12 selected genes. Our analysis provides novel information that can help delineate the molecular mechanism and components of plant responses to effectors, which will be useful for the development of resistant crops.

Pollen Coat Proteomes of Arabidopsis thaliana, Arabidopsis lyrata, and Brassica oleracea Reveal Remarkable Diversity of Small Cysteine-Rich Proteins at the Pollen-Stigma Interface

The pollen coat is the outermost domain of the pollen grain and is largely derived from the anther tapetum, which is a secretory tissue that degenerates late in pollen development. By being localised at the interface of the pollen-stigma interaction, the pollen coat plays a central role in mediating early pollination events, including molecular recognition. Amongst species of the Brassicaceae, a growing body of data has revealed that the pollen coat carries a range of proteins, with a number of small cysteine-rich proteins (CRPs) being identified as important regulators of the pollen-stigma interaction. By utilising a state-of-the-art liquid chromatography/tandem mass spectrometry (LC-MS/MS) approach, rich pollen coat proteomic profiles were obtained for Arabidopsis thaliana, Arabidopsis lyrata, and Brassica oleracea, which greatly extended previous datasets. All three proteomes revealed a strikingly large number of small CRPs that were not previously reported as pollen coat components. The profiling also uncovered a wide range of other protein families, many of which were enriched in the pollen coat proteomes and had functions associated with signal transduction, cell walls, lipid metabolism and defence. These proteomes provide an excellent source of molecular targets for future investigations into the pollen-stigma interaction and its potential evolutionary links to plant-pathogen interactions.

Do egg cell‐secreted aspartic proteases promote gamete attachment?

During the double fertilization process of flowering plants (angiosperms), pollen tubes transport a pair of immobile sperm cells towards and inside the embryo sac for fusion with egg and central cell, respectively. Male-female gamete interactions during fertilization are critical for reproductive success and seed development, and thus have interested biologists since decades. Compared to animals, little is known about gamete interaction mechanisms in angiosperms. This article is protected by copyright. All rights reserved.

The role of metallothionein in breast cancer

Metallothioneins (MTs) are a family of small cysteine-rich proteins involved in many physiological and pathological processes. Genes that encode the four isoforms of metallothioneins (MT1-M4) are located on chromosome 16q13. Strucutures of these four isoforms allow metallothioneins their various biological functions. Many studies have shown that MT plays an important role in carcinogenesis, tumour growth, its progression from local to metastatic disease and may contribute to resistence to chemotherapy and radiotherapy. Due to the fact that breast cancer is one of the leading causes of death in women worldwide it is important to better understand the biology of breast cancer. So, findings of MT could evenutally help as a prognostic tool and could lead to a possible new specific anti-cancer treatment.

Homo- and Heteronuclear Group 12 Metallothionein Type B Cluster Analogs: Synthesis, Structure, 1H NMR and ESI-MS.

Metallothioneins (MTs) are a ubiquitous class of small cysteine-rich metal-binding proteins involved in metal homeostasis and detoxification with highly versatile metal binding properties. Despite the long-standing association of MT with M3S3 and M4S5 metal clusters, synthetic complexes with these core architectures are exceptionally rare. Here, we demonstrate an approach to synthesizing and characterizing aggregates of group 12 metal ions with monocyclic M3S3 cores in acetonitrile solution without the protection of a protein. Multidentate monothiol ligand N,N-bis(2-pyridylmethyl)-2-aminoethanethiol (L1H) provided [Cd3(L1)3](ClO4)3 (1), the first structurally characterized nonproteinaceous aggregate with a metallothionein-like monocyclic Cd3S3 core. In addition, [Zn3(L1)3](ClO4)3·4CH3CN (2·4CH3CN) was characterized by X-ray crystallography. The complex cations of 1 and 2 had comparable structures despite being nonisomorphic. Variable temperature and concentration 1H NMR were used to investigate aggregation equilibria of 1, 2, and a precipitate with composition "Hg(L1)(ClO4)" (3). Cryogenic 1H NMR studies of 3 revealed a J(199Hg1H) coupling constant pattern consistent with an aggregate possessing a cyclic core. ESI-MS was used for gas-phase characterization of 1-3, as well as mixed-metal [M2M'(L1)3(ClO4)2]+ ions prepared in situ by pairwise acetonitrile solution combinations of the group 12 complexes of L1. Access to synthetic variants of metallothionein-like group 12 aggregates provides an additional approach to understanding their behavior.

Integrative transcriptome analysis revealed the pathogenic molecular basis of Rhizoctonia solani AG-3 TB at three progressive stages of infection.

Rhizoctonia solani has a broad host range and results in significant losses in agricultural production. Here, an integrated transcriptomic analysis was performed to reveal the critical genes responsible for the pathogenesis of R. solani AG-3 TB on Nicotiana tabacum at different infection stages. The results showed that various differential expressed genes (DEGs) were enriched in fatty acid metabolism, amino sugar, carbon metabolism, and cellular carbohydrate biosynthetic process at the early (6–12 hpi), middle (24–36 hpi), and late stage (48–72 hpi) of infection. Specifically, several critical genes such as shikimate kinase that were involved in the biosynthesis of an important fungal toxin, phenylacetic acid (PAA) showed markedly increase at 24 hpi. Additionally, the genes expression levels of carbohydrate-active enzymes (CAZymes) and cell wall degrading enzymes (CWDEs) were significantly increased at the late infection stage. Furthermore, we identified 807 potential secreted proteins and 78 small cysteine-rich proteins, which may function as fungal effectors and involved in the pathogenicity. These results provide valuable insights into critical and potential genes as well as the pathways involved in the pathogenesis of R. solani AG-3 TB.

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.

Genome-Wide Identification and Expression Analysis of nsLTP Gene Family in Rapeseed (Brassica napus) Reveals Their Critical Roles in Biotic and Abiotic Stress Responses.

Non-specific lipid transfer proteins (nsLTPs) are small cysteine-rich basic proteins which play essential roles in plant growth, development and abiotic/biotic stress response. However, there is limited information about the nsLTP gene (BnLTP) family in rapeseed (Brassica napus). In this study, 283 BnLTP genes were identified in rapeseed, which were distributed randomly in 19 chromosomes of rapeseed. Phylogenetic analysis showed that BnLTP proteins were divided into seven groups. Exon/intron structure and MEME motifs both remained highly conserved in each BnLTP group. Segmental duplication and hybridization of rapeseed’s two sub-genomes mainly contributed to the expansion of the BnLTP gene family. Various potential cis-elements that respond to plant growth, development, biotic/abiotic stresses, and phytohormone signals existed in BnLTP gene promoters. Transcriptome analysis showed that BnLTP genes were expressed in various tissues/organs with different levels and were also involved in the response to heat, drought, NaCl, cold, IAA and ABA stresses, as well as the treatment of fungal pathogens (Sclerotinia sclerotiorum and Leptosphaeria maculans). The qRT-PCR assay validated the results of RNA-seq expression analysis of two top Sclerotinia-responsive BnLTP genes, BnLTP129 and BnLTP161. Moreover, batches of BnLTPs might be regulated by BnTT1 and BnbZIP67 to play roles in the development, metabolism or adaptability of the seed coat and embryo in rapeseed. This work provides an important basis for further functional study of the BnLTP genes in rapeseed quality improvement and stress resistance.

Newly Discovered Peptides from the Coral Heliofungia actiniformis Show Structural and Functional Diversity.

Scleractinian corals are crucially important to the health of some of the world's most biodiverse, productive, and economically important marine habitats. Despite this importance, analysis of coral peptidomes is still in its infancy. Here we show that the tentacle extract from the stony coral Heliofungia actiniformis is rich in peptides with diverse and novel structures. We have characterized the sequences and three-dimensional structures of four new peptides, three of which have no known homologues. We show that a 2 kDa peptide, Hact-2, promotes significant cell proliferation on human cells and speculate this peptide may be involved in the remarkable regenerative capacity of corals. We found a 3 kDa peptide, Hact-3, encoded within a fascin-like domain, and homologues of Hact-3 are present in the genomes of other coral species. Two additional peptides, Hact-4 and Hact-SCRiP1, with limited sequence similarity, both contain a beta-defensin-like fold and highlight a structural link with the small cysteine-rich proteins (SCRiP) family of proteins found predominantly in corals. Our results provide a first glimpse into the remarkable and unexplored structural diversity of coral peptides, providing insight into their diversity and putative functions and, given the ancient lineage of corals, potential insight into the evolution of structural motifs.

The Small Secreted Protein FoSsp1 Elicits Plant Defenses and Negatively Regulates Pathogenesis in Fusarium oxysporum f. sp. cubense (Foc4).

Fusarium wilt of banana (Musa spp.), a typical vascular wilt disease caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense race 4 (Foc4), seriously threatens banana production worldwide. Pathogens, including vascular wilt fungi, secrete small cysteine-rich proteins during colonization. Some of these proteins are required for pathogenicity. In this study, 106 small secretory proteins that contain a classic N-terminal signal peptide were identified using bioinformatic methods in Foc4. Among them, 11 proteins were selected to show transient expressions in tobacco. Interestingly, transient expression of FoSsp1 in tobacco, an uncharacterized protein (of 145 aa), induced necrotic cell death reactive oxygen burst, and callous deposition. Furthermore, the expression of FoSSP1 in Foc4 wild type (WT) was up-regulated during the stage of banana roots colonization. A split-marker approach was used to knock out FoSSP1 in the Foc4 WT strain. Compared with the WT, the deletion mutant Fossp1 was normal in growth rate but increased in conidiation and virulence. RT-qPCR analysis showed that the expression of four conidiation regulator genes in the Fossp1 deletion mutant was significantly decreased compared to the WT strain. In addition, the expression of four pathogenesis-related genes of bananas infected with Fossp1 deletion mutant was down-regulated in comparison with that of the WT. In summary, these results suggested that FoSSP1 is a putative elicitor that negatively regulates conidiation and pathogenicity in Foc4.

Verticillium dahliae CFEM proteins manipulate host immunity and differentially contribute to virulence

BackgroundVerticillium dahliae is a fungal pathogen that causes a vascular wilt on many economically important crops. Common fungal extracellular membrane (CFEM) domain proteins including secreted types have been implicated in virulence, but their roles in this pathogen are still unknown.ResultsNine secreted small cysteine-rich proteins (VdSCPs) with CFEM domains were identified by bioinformatic analyses and their differential suppression of host immune responses were evaluated. Two of these proteins, VdSCP76 and VdSCP77, localized to the plant plasma membrane owing to their signal peptides and mediated broad-spectrum suppression of all immune responses induced by typical effectors. Deletion of either VdSCP76 or VdSCP77 significantly reduced the virulence of V. dahliae on cotton. Furthermore, VdSCP76 and VdSCP77 suppressed host immunity through the potential iron binding site conserved in CFEM family members, characterized by an aspartic acid residue in seven VdSCPs (Asp-type) in contrast with an asparagine residue (Asn-type) in VdSCP76 and VdSCP77. V. dahliae isolates carrying the Asn-type CFEM members were more virulent on cotton than those carrying the Asp-type.ConclusionsIn the iron-insufficient xylem, V. dahliae is likely to employ the Asp-type CFEM members to chelate iron, and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants.

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).