Putative Effector Proteins Research Articles

Mycobacterial PE/PPE proteins function as “personal protective equipment” against host defenses

Mycobacterium tuberculosis (Mtb) is the deadliest bacterial infection worldwide, but many molecular details of how it interacts with the innate immune system remain obscure. In particular, although Mtb secretes a large number of putative effector proteins, a relatively small number have assigned functions in facilitating host-pathogen interactions. One particularly large family of secreted mycobacterial proteins that remains poorly understood is the PE/PPE proteins. Despite numerous lines of evidence for potential roles in virulence and in mediating host-pathogen interactions, only a small fraction of these 170+ proteins have been well characterized. However, this large family of proteins is likely key for understanding how Mtb subverts immune responses, manipulates host cell biology, and establishes a successful infection. Here, we highlight examples of PE/PPEs that have well-defined effects on cell intrinsic pathways in macrophages during mycobacterial infection. Examples include PPE2, which blunts production of reactive oxygen species and nitric oxide; PE_PGRS33, which facilitates bacterial uptake; PE_PGRS29, which directly binds ubiquitin to promote host autophagy and limit pathologic inflammation; MirA, which facilitates actin tail formation to promote cell-to-cell spread; and others. Understanding the full spectrum of PE/PPE functions is critical for understanding Mtb pathogenesis and for developing new strategies to combat the worldwide TB pandemic. Advancing the lagging research efforts characterizing this mysterious family of effector proteins is critical for the TB field.

The soil-borne white root rot pathogen Rosellinia necatrix expresses antimicrobial proteins during host colonization.

Rosellinia necatrix is a prevalent soil-borne plant-pathogenic fungus that is the causal agent of white root rot disease in a broad range of host plants. The limited availability of genomic resources for R. necatrix has complicated a thorough understanding of its infection biology. Here, we sequenced nine R. necatrix strains with Oxford Nanopore sequencing technology, and with DNA proximity ligation we generated a gapless assembly of one of the genomes into ten chromosomes. Whereas many filamentous pathogens display a so-called two-speed genome with more dynamic and more conserved compartments, the R. necatrix genome does not display such genome compartmentalization. It has recently been proposed that fungal plant pathogens may employ effectors with antimicrobial activity to manipulate the host microbiota to promote infection. In the predicted secretome of R. necatrix, 26 putative antimicrobial effector proteins were identified, nine of which are expressed during plant colonization. Two of the candidates were tested, both of which were found to possess selective antimicrobial activity. Intriguingly, some of the inhibited bacteria are antagonists of R. necatrix growth in vitro and can alleviate R. necatrix infection on cotton plants. Collectively, our data show that R. necatrix encodes antimicrobials that are expressed during host colonization and that may contribute to modulation of host-associated microbiota to stimulate disease development.

Uncovering the structural stability of Magnaporthe oryzae effectors: a secretome-wide in silico analysis

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae, is a deadly disease and a major threat to global food security. The pathogen secretes small proteinaceous effectors, virulence factors, inside the host to manipulate and perturb the host immune system, allowing the pathogen to colonize and establish a successful infection. While the molecular functions of several effectors are characterized, very little is known about the structural stability of these effectors. We analyzed a total of 554 small secretory proteins (SSPs) from the M. oryzae secretome to decipher key features of intrinsic disorder (ID) and the structural dynamics of the selected putative effectors through thorough and systematic in silico studies. Our results suggest that out of the total SSPs, 66% were predicted as effector proteins, released either into the apoplast or cytoplasm of the host cell. Of these, 68% were found to be intrinsically disordered effector proteins (IDEPs). Among the six distinct classes of disordered effectors, we observed peculiar relationships between the localization of several effectors in the apoplast or cytoplasm and the degree of disorder. We determined the degree of structural disorder and its impact on protein foldability across all the putative small secretory effector proteins from the blast pathogen, further validated by molecular dynamics simulation studies. This study provides definite clues toward unraveling the mystery behind the importance of structural distortions in effectors and their impact on plant-pathogen interactions. The study of these dynamical segments may help identify new effectors as well. Communicated by Ramaswamy H. Sarma

Whole genome sequencing and analysis of multiple isolates of Ceratocystis destructans, the causal agent of Ceratocystis canker of almond in California

Ceratocystis canker caused by Ceratocystis destructans is a severe disease of almond, reducing the longevity and productivity of infected trees. Once the disease has established in an individual tree, there is no cure, and management efforts are often limited to removing the infected area of cankers. In this study, we present the genome assemblies of five C. destructans isolates isolated from symptomatic almond trees. The genomes were assembled into a genome size of 27.2 ± 0.9 Mbp with an average of 6924 ± 135 protein-coding genes and an average GC content of 48.8 ± 0.02%. We concentrated our efforts on identifying putative virulence factors of canker pathogens. Analysis of the secreted carbohydrate-active enzymes showed that the genomes harbored 83.4 ± 1.8 secreted CAZymes. The secreted CAZymes covered all the known categories of CAZymes. AntiSMASH revealed that the genomes had at least 7 biosynthetic gene clusters, with one of the non-ribosomal peptide synthases encoding dimethylcoprogen, a conserved virulence determinant of plant pathogenic ascomycetes. From the predicted proteome, we also annotated cytochrome P450 monooxygenases, and transporters, these are well-established virulence determinants of canker pathogens. Moreover, we managed to identify 57.4 ± 2.1 putative effector proteins. Gene Ontology (GO) annotation was applied to compare gene content with two closely related species C. fimbriata, and C. albifundus. This study provides the first genome assemblies for C. destructans, expanding genomic resources for an important almond canker pathogen. The acquired knowledge provides a foundation for further advanced studies, such as molecular interactions with the host, which is critical for breeding for resistance.

Bacterial secretion systems contribute to rapid tissue decay in button mushroom soft rot disease.

The soft rot pathogen Janthinobacterium agaricidamnosum causes devastating damage to button mushrooms (Agaricus bisporus), one of the most cultivated and commercially relevant mushrooms. We previously discovered that this pathogen releases the membrane-disrupting lipopeptide jagaricin. This bacterial toxin, however, could not solely explain the rapid decay of mushroom fruiting bodies, indicating that J. agaricidamnosum implements a more sophisticated infection strategy. In this study, we show that secretion systems play a crucial role in soft rot disease. By mining the genome of J. agaricidamnosum, we identified gene clusters encoding a type I (T1SS), a type II (T2SS), a type III (T3SS), and two type VI secretion systems (T6SSs). We targeted the T2SS and T3SS for gene inactivation studies, and subsequent bioassays implicated both in soft rot disease. Furthermore, through a combination of comparative secretome analysis and activity-guided fractionation, we identified a number of secreted lytic enzymes responsible for mushroom damage. Our findings regarding the contribution of secretion systems to the disease process expand the current knowledge of bacterial soft rot pathogens and represent a significant stride toward identifying targets for their disarmament with secretion system inhibitors. IMPORTANCE The button mushroom (Agaricus bisporus) is the most popular edible mushroom in the Western world. However, mushroom crops can fall victim to serious bacterial diseases that are a major threat to the mushroom industry, among them being soft rot disease caused by Janthinobacterium agaricidamnosum. Here, we show that the rapid dissolution of mushroom fruiting bodies after bacterial invasion is due to degradative enzymes and putative effector proteins secreted via the type II secretion system (T2SS) and the type III secretion system (T3SS), respectively. The ability to degrade mushroom tissue is significantly attenuated in secretion-deficient mutants, which establishes that secretion systems are key factors in mushroom soft rot disease. This insight is of both ecological and agricultural relevance by shedding light on the disease processes behind a pathogenic bacterial-fungal interaction which, in turn, serves as a starting point for the development of secretion system inhibitors to control disease progression.

Prediction of Effector Proteins from Trichoderma longibrachiatum Through Transcriptome Sequencing.

Trichoderma longibrachiatum SMF2 is an important biocontrol strain isolated by our group that can promote plant growth and induce plant disease resistance. To further study its biocontrol mechanism, the effector proteins secreted by T. longibrachiatum SMF2 were analyzed through bioinformatics and transcriptome sequencing. Overall, 478 secretory proteins produced by T. longibrachiatum were identified, of which 272 were upregulated after treatment with plants. Functional annotation showed that 36 secretory proteins were homologous with different groups of effectors from pathogenic microorganisms. Moreover, the quantitative PCR results of six putative effector proteins were consistent with those of transcriptome sequencing. Taken together, these findings indicate that the secretory proteins secreted by T. longibrachiatum SMF2 may act as effectors to facilitate its own growth and colonization or to induce plant immunity response.

Comparative genomic assessment of members of genus Tenacibaculum: an exploratory study.

Tenacibaculosis is an ulcerative skin disorder that affects finfish. It is caused by members of the genus Tenacibaculum, resulting in eccentric behavioural changes, including anorexia, lethargy, and abnormal swimming patterns that often result in mortality. Currently, species suspected of causing fish mortality include T. ovolyticum, T. gallaicum, T. discolor, T. finnmarkense, T. mesophilum, T. soleae, T. dicentrarchi, and T. maritimum. However, pathogenic members and the mechanisms involved in disease causation, progression, and transmission are limited due to the inadequate sequencing efforts in the past decade. In this study, we use a comparative genomics approach to investigate the characteristic features of 26 publicly available genomes of Tenacibaculum and report our observations. We propose the reclassification of "T. litoreum HSC 22" to the singaporense species and assignment of "T. sp. 4G03" to the species discolor (species with quotation marks have not been appropriately named). We also report the co-occurrence of several antimicrobial resistance/virulence genes and genes private to a few members. Finally, we mine several non-B DNA forming regions, operons, tandem repeats, high-confidence putative effector proteins, and sortase that might play a pivotal role in bacterial evolution, transcription, and pathogenesis.

The effector Fg62 contributes to Fusarium graminearum virulence and induces plant cell death

Although the functions of most protein effectors secreted by fungi are not predictable, they are known to modulate host immunity and facilitate infection. Fusarium graminearum is one of the 10 most abundant plant pathogenic fungi worldwide. To date, few effector proteins secreted by F. graminearum have been identified. In this study, we screened a putative effector protein Fg62 from proteins that contain signal peptides and unknown functional domains in F. graminearum secretome. Fg62 expression was highly upregulated during the early stages of F. graminearum infection, and its deletion reduced F. graminearum virulence in wheat and soybean. Transient expression of Fg62 or the recombinant protein led to plant cell death in Nicotiana benthamiana, and the signal peptide of Fg62 was required for cell death activation. Fg62 homologs are distributed in two species of the F. sambucinum species complex, which are also able to induce cell death in N. benthamiana. Fg62 activated plant immunity by increasing the expression of defense-related genes, and the recombinant Fg62 protein induced plant resistance to various pathogens. Overall, our results revealed that the extracellular effector Fg62 contributes to both pathogen virulence and plant immunity induction, providing new avenues for the development of environmentally friendly crop disease control strategies utilizing nonpolluting immune-inducing factors.

Effector-mediated partial and nonhost disease resistance in wheat.

Effector-mediated partial and nonhost disease resistance in wheat.

Genome-Wide Characterization of Effector Protein-Encoding Genes in Sclerospora graminicola and Its Validation in Response to Pearl Millet Downy Mildew Disease Stress.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is the essential food crop for over ninety million people living in drier parts of India and South Africa. Pearl millet crop production is harshly hindered by numerous biotic stresses. Sclerospora graminicola causes downy mildew disease in pearl millet. Effectors are the proteins secreted by several fungi and bacteria that manipulate the host cell structure and function. This current study aims to identify genes encoding effector proteins from the S. graminicola genome and validate them through molecular techniques. In silico analyses were employed for candidate effector prediction. A total of 845 secretory transmembrane proteins were predicted, out of which 35 proteins carrying LxLFLAK (Leucine-any amino acid-Phenylalanine-Leucine-Alanine-Lysine) motif were crinkler, 52 RxLR (Arginine, any amino acid, Leucine, Arginine), and 17 RxLR-dEER putative effector proteins. Gene validation analysis of 17 RxLR-dEER effector protein-producing genes was carried out, of which 5genes were amplified on the gel. These novel gene sequences were submitted to NCBI. This study is the first report on the identification and characterization of effector genes in Sclerospora graminicola. This dataset will aid in the integration of effector classes that act independently, paving the way to investigate how pearl millet responds to effector protein interactions. These results will assist in identifying functional effector proteins involving the omic approach using newer bioinformatics tools to protect pearl millet plants against downy mildew stress. Considered together, the identified effector protein-encoding functional genes can be utilized in screening oomycetes downy mildew diseases in other crops across the globe.

Cytoplasmic contractile injection systems mediate cell death in Streptomyces

Contractile injection systems (CIS) are bacteriophage tail-like structures that mediate bacterial cell–cell interactions. While CIS are highly abundant across diverse bacterial phyla, representative gene clusters in Gram-positive organisms remain poorly studied. Here we characterize a CIS in the Gram-positive multicellular model organism Streptomyces coelicolor and show that, in contrast to most other CIS, S. coelicolor CIS (CISSc) mediate cell death in response to stress and impact cellular development. CISSc are expressed in the cytoplasm of vegetative hyphae and are not released into the medium. Our cryo-electron microscopy structure enabled the engineering of non-contractile and fluorescently tagged CISSc assemblies. Cryo-electron tomography showed that CISSc contraction is linked to reduced cellular integrity. Fluorescence light microscopy furthermore revealed that functional CISSc mediate cell death upon encountering different types of stress. The absence of functional CISSc had an impact on hyphal differentiation and secondary metabolite production. Finally, we identified three putative effector proteins, which when absent, phenocopied other CISSc mutants. Our results provide new functional insights into CIS in Gram-positive organisms and a framework for studying novel intracellular roles, including regulated cell death and life-cycle progression in multicellular bacteria.

Transcriptomic and proteomic analyses provide insights into host adaptation of a bamboo-feeding aphid.

Salivary glands and their secreted proteins play an important role in the feeding process of sap-sucking aphids. The determination of saliva composition is an important step in understanding host plant adaptation of aphids. Pseudoregma bambucicola is a severe bamboo pest in subtropical areas and the only aphid species that can exclusively feed on hard stalks of bamboos. How this species can penetrate and degrade hard bamboo cell walls and utilize a very specialized niche are important unanswered questions. In this study, comprehensive analyses based on transcriptome sequencing, RT-qPCR, liquid chromatography-tandem spectrometry (LC-MS/MS) and bioinformatics were conducted on dissected salivary glands and secreted saliva of P. bambucicola to characterize the overall gene expression and salivary protein composition, and to identify putative effector proteins important for aphid-plant interactions. Some secretory proteins homologous to known aphid effectors important for aphid-plant interactions, such as digestive enzymes, detoxifying and antioxidant enzymes and some effectors modulating plant defenses, are also detected in salivary gland transcriptome and salivary gland and/or saliva secretomes in P. bambucicola. This indicates that these effectors are probably be essential for enabling P. bambucicola feeding on bamboo host. Although several plant cell wall degrading enzymes (PCWDEs) can be identified from transcriptome, most of the enzymes identified in salivary glands showed low expression levels and they only represent a small fraction of the complete set of enzymes for degrading cellulose and hemicellulose. In addition, our data show that P. bambucicola has no its own ability to produce pectinases. Overall, our analyses indicate that P. bambucicola may lose its own ability to express and secrete key PCWDEs, and its adaptation to unique feeding habit may depend on its symbiotic bacteria.

The inside scoop: Comparative genomics of two intranuclear bacteria, "Candidatus Berkiella cookevillensis" and "Candidatus Berkiella aquae".

"Candidatus Berkiella cookevillensis" (strain CC99) and "Candidatus Berkiella aquae" (strain HT99), belonging to the Coxiellaceae family, are gram-negative bacteria isolated from amoebae in biofilms present in human-constructed water systems. Both bacteria are obligately intracellular, requiring host cells for growth and replication. The intracellular bacteria-containing vacuoles of both bacteria closely associate with or enter the nuclei of their host cells. In this study, we analyzed the genome sequences of CC99 and HT99 to better understand their biology and intracellular lifestyles. The CC99 genome has a size of 2.9Mb (37.9% GC) and contains 2,651 protein-encoding genes (PEGs) while the HT99 genome has a size of 3.6Mb (39.4% GC) and contains 3,238 PEGs. Both bacteria encode high proportions of hypothetical proteins (CC99: 46.5%; HT99: 51.3%). The central metabolic pathways of both bacteria appear largely intact. Genes for enzymes involved in the glycolytic pathway, the non-oxidative branch of the phosphate pathway, the tricarboxylic acid pathway, and the respiratory chain were present. Both bacteria, however, are missing genes for the synthesis of several amino acids, suggesting reliance on their host for amino acids and intermediates. Genes for type I and type IV (dot/icm) secretion systems as well as type IV pili were identified in both bacteria. Moreover, both bacteria contain genes encoding large numbers of putative effector proteins, including several with eukaryotic-like domains such as, ankyrin repeats, tetratricopeptide repeats, and leucine-rich repeats, characteristic of other intracellular bacteria.

The Population of Fusarium oxysporum f. sp. cubense in Brazil Is Not Structured by Vegetative Compatibility Group or by Geographic Origin.

Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f. sp. cubense (Foc), is considered one of the most destructive diseases of bananas in Brazil. In this study, a collection of 194 monosporic isolates from several banana-producing regions located in different climatic zones along a south-to-north transect in Brazil was formed to assess the genetic structure of the population of Foc. The isolates underwent pathogenicity tests, PCR diagnosis for the detection of tropical race 4, and screening of SIX homolog genes that produce putative effector proteins. The vegetative compatibility group (VCG) of 119 isolates was determined by pairing against 17 internationally known VCG-tester strains. A group of 158 isolates was selected for simple sequence repeat (SSR) genotyping. There was moderate diversity of Foc in Brazil. Eight VCGs were identified: 0120, 0122, 0124, 0125, 0128, 01215, 01220, and 01222, of which 78% of isolates belong to a single VCG, whereas 22% of isolates are assigned to multiple VCGs, belonging to complexes of VCGs. The distribution of VCGs is uneven and independent of the banana genotype. The isolates of a VCG shared a similar profile of SIX homologs, but there was no association with geographic region. Four SSR loci were polymorphic, and, on average, 7.5 alleles were detected per locus. Thirty-five multilocus genotypes (MLGs) were identified. There was no association between VCG and MLGs, and no genetic structure of the population of Foc in Brazil was detected.

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, indicating an involvement in the fungal infection process.

FoCupin1, a Cupin_1 domain-containing protein, is necessary for the virulence of Fusarium oxysporum f. sp. cubense tropical race 4.

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is an important soilborne fungal pathogen that causes the most devastating banana disease. Effectors secreted by microbes contribute to pathogen virulence on host plants in plant-microbe interactions. However, functions of Foc TR4 effectors remain largely unexplored. In this study, we characterized a novel cupin_1 domain-containing protein (FoCupin1) from Foc TR4. Sequence analysis indicated that the homologous proteins of FoCupin1 in phytopathogenic fungi were evolutionarily conserved. Furthermore, FoCupin1 could suppress BAX-mediated cell death and significantly downregulate the expression of defense-related genes in tobacco by using the Agrobacterium-mediated transient expression system. FoCupin1 was highly induced in the early stage of Foc TR4 infection. The deletion of FoCupin1 gene did not affect Foc TR4 growth and conidiation. However, FoCupin1 deletion significantly reduced Foc TR4 virulence on banana plants, which was further confirmed by biomass assay. The expression of the defense-related genes in banana was significantly induced after inoculation with FoCupin1 mutants. These results collectively indicate FoCupin1 is a putative effector protein that plays an essential role in Foc TR4 pathogenicity. These findings suggest a novel role for cupin_1 domain-containing proteins and deepen our understanding of effector-mediated Foc TR4 pathogenesis.

The Complete Genome of the "Flavescence Dorée" Phytoplasma Reveals Characteristics of Low Genome Plasticity.

Simple SummaryPhytoplasmas are non-cultivable organisms for which biological and molecular characterization is a challenge. Despite the development of new sequencing technologies, few high- quality phytoplasma genomes are available. This research describes the genome sequencing of the phytoplasma involved in grapevine Flavescence dorée, a devastating disease in European vineyards. Analysis of the gene content and comparison with other representative phytoplasmas revealed specific characteristics of the “Flavescence dorée” phytoplasma chromosome. Their implications for the development of the disease are presented and discussed.Members of the genus ‘Candidatus Phytoplasma’ are obligate intracellular bacteria restricted to phloem sieve elements and are able to colonize several tissues and the hemolymph in their insect vectors. The current unfeasibility of axenic culture and the low complexity of genomic sequences are obstacles in assembling complete chromosomes. Here, a method combining pathogen DNA enrichment from infected insects and dual deep-sequencing technologies was used to obtain the complete genome of a phytoplasma causing Grapevine Flavescence dorée. The de novo assembly generated a circular chromosome of 654,223 bp containing 506 protein-coding genes. Quality assessment of the draft showed a high degree of completeness. Comparative analysis with other phytoplasmas revealed the absence of potential mobile units and a reduced amount of putative phage-derived segments, suggesting a low genome plasticity. Phylogenetic analyses identified Candidatus Phytoplasma ziziphi as the closest fully sequenced relative. The “Flavescence dorée” phytoplasma strain CH genome also encoded for several putative effector proteins potentially playing a role in pathogen virulence. The availability of this genome provides the basis for the study of the pathogenicity mechanisms and evolution of the Flavescence dorée phytoplasma.

Phytoplasma effector Zaofeng6 induces shoot proliferation by decreasing the expression of ZjTCP7 in Ziziphus jujuba

The jujube witches' broom (JWB) phytoplasma is associated with witches' broom, dwarfism, and smaller leaves in jujube, resulting in yield losses. In this study, eight putative JWB effector proteins were identified from potential mobile units of the JWB genome. Among them, Zaofeng6 induced witches' broom symptoms in Arabidopsis and jujube. Zaofeng6-overexpressing Arabidopsis and unrooted jujube transformants displayed witches' broom-like shoot proliferation. Transient expression of Zaofeng6 induced hypersensitive response like cell death and expression of hypersensitive response marker genes, like harpin-induced gene 1 (H1N1), and the pathogenesis-related genes PR1, PR2, and PR3 in transformed Nicotiana benthamiana leaves, suggesting that Zaofeng6 could be a virulence effector. Yeast two-hybrid library screening and bimolecular fluorescence complementation confirmed that Zaofeng6 interacts with ZjTCP7 through its first two α-helix domains in the cell nuclei. ZjTCP7 mRNA and protein abundance decreased in Zaofeng6 transgenic jujube seedlings. The expression of some genes in the strigolactone signaling pathway (ZjCCD7, ZjCCD8, and CYP711A1) were down-regulated in jujube shoots overexpressing Zaofeng6 and in zjtcp7 CRISPR/Cas9 mutants. Zaofeng6 induces shoot proliferation through decreased expression of ZjTCP7 at the transcriptional and translational levels.

Characterization of a Secretory YML079-like Cupin Protein That Contributes to Sclerotinia sclerotiorum Pathogenicity.

Sclerotinia sclerotiorum causes devastating diseases in many agriculturally important crops, including oilseed rape and sunflower. However, the mechanisms of Sclerotinia sclerotiorum pathogenesis remain poorly understood. In this study, we characterized a YML079-like cupin protein (SsYCP1) from Sclerotinia sclerotiorum. We showed that SsYCP1 is strongly expressed and secreted during Sclerotinia sclerotiorum infection. Sclerotinia sclerotiorum infection was promoted by SsYCP1 overexpression and inhibited by silencing this gene with synthetic double-stranded RNA. These results collectively indicate SsYCP1 as a putative effector protein that contributes to Sclerotinia sclerotiorum pathogenicity. These findings extend our understanding of effector-mediated Sclerotinia sclerotiorum pathogenesis and suggest a novel role for YML079-like cupin proteins in plant–pathogen interactions.

A novel effector, CsSp1, from Bipolaris sorokiniana, is essential for colonization in wheat and is also involved in triggering host immunity.

The hemibiotrophic pathogen Bipolaris sorokiniana causes root rot, leaf blotching, and black embryos in wheat and barley worldwide, resulting in significant yield and quality reductions. However, the mechanism underlying the host–pathogen interactions between B. sorokiniana and wheat or barley remains unknown. The B. sorokiniana genome encodes a large number of uncharacterized putative effector proteins. In this study, we identified a putative secreted protein, CsSp1, with a classic N‐terminal signal peptide, that is induced during early infection. A split‐marker approach was used to knock out CsSP1 in the Lankao 9‐3 strain. Compared with the wild type, the deletion mutant ∆Cssp1 displayed less radial growth on potato dextrose agar plates and produced fewer spores, and complementary transformation completely restored the phenotype of the deletion mutant to that of the wild type. The pathogenicity of the deletion mutant in wheat was attenuated even though appressoria still penetrated the host. Additionally, the infectious hyphae in the deletion mutant became swollen and exhibited reduced growth in plant cells. The signal peptide of CsSp1 was functionally verified through a yeast YTK12 secretion system. Transient expression of CsSp1 in Nicotiana benthamiana inhibited lesion formation caused by Phytophthora capsici. Moreover, CsSp1 localized in the nucleus and cytoplasm of plant cells. In B. sorokiniana‐infected wheat leaves, the salicylic acid‐regulated genes TaPAL, TaPR1, and TaPR2 were down‐regulated in the ∆Cssp1 strain compared with the wild‐type strain under the same conditions. Therefore, CsSp1 is a virulence effector and is involved in triggering host immunity.