Small Secreted Protein Research Articles

Genetics and breeding for resistance against four leaf spot diseases in wheat (Triticum aestivum L.).

In wheat, major yield losses are caused by a variety of diseases including rusts, spike diseases, leaf spot and root diseases. The genetics of resistance against all these diseases have been studied in great detail and utilized for breeding resistant cultivars. The resistance against leaf spot diseases caused by each individual necrotroph/hemi-biotroph involves a complex system involving resistance (R) genes, sensitivity (S) genes, small secreted protein (SSP) genes and quantitative resistance loci (QRLs). This review deals with resistance for the following four-leaf spot diseases: (i) Septoria nodorum blotch (SNB) caused by Parastagonospora nodorum; (ii) Tan spot (TS) caused by Pyrenophora tritici-repentis; (iii) Spot blotch (SB) caused by Bipolaris sorokiniana and (iv) Septoria tritici blotch (STB) caused by Zymoseptoria tritici.

Pathogenic Adaptations Revealed by Comparative Genome Analyses of Two Colletotrichum spp., the Causal Agent of Anthracnose in Rubber Tree.

Colletotrichum siamense and Colletotrichum australisinense cause Colletotrichum leaf disease that differ in their symptoms in rubber tree (Hevea brasiliensis), and pathogenicity of these two fungal species is also not identical on different cultivars of rubber tree. This divergence is often attributed to pathogen virulence factors, namely carbohydrate-active enzymes (CAZymes), secondary metabolites (SM), and small-secreted protein (SSP) effectors. The draft genome assembly and functional annotation of potential pathogenicity genes of both species obtained here provide an important and timely genomic resource for better understanding the biology and lifestyle of Colletotrichum spp. This should pave the way for designing more efficient disease control strategies in plantations of rubber tree. In this study, the genes associated with these categories were manually annotated in the genomes of C. australisinense GX1655 and C. siamense HBCG01. Comparative genomic analyses were performed to address the evolutionary relationships among these gene families in the two species. First, the size of genome assembly, number of predicted genes, and some of the functional categories differed significantly between the two congeners. Second, from the comparative genomic analyses, we identified some specific genes, certain higher abundance of gene families associated with CAZymes, CYP450, and SM in the genome of C. siamense, and Nep1-like proteins (NLP) in the genome of C. australisinense.

The small secreted effector protein MiSSP7.6 of Laccaria bicolor is required for the establishment of ectomycorrhizal symbiosis.

To establish and maintain a symbiotic relationship, the ectomycorrhizal fungus Laccaria bicolor releases mycorrhiza-induced small secreted proteins (MiSSPs) into host roots. Here, we have functionally characterized the MYCORRHIZA-iNDUCED SMALL SECRETED PROTEIN OF 7.6 kDa (MiSSP7.6) from L. bicolor by assessing its induced expression in ectomycorrhizae, silencing its expression by RNAi, and tracking in planta subcellular localization of its protein product. We also carried out yeast two-hybrid assays and bimolecular fluorescence complementation analysis to identify possible protein targets of the MiSSP7.6 effector in Populus roots. We showed that MiSSP7.6 expression is upregulated in ectomycorrhizal rootlets and associated extramatrical mycelium during the late stage of symbiosis development. RNAi mutants with a decreased MiSSP7.6 expression have a lower mycorrhization rate, suggesting a key role in the establishment of the symbiosis with plants. MiSSP7.6 is secreted, and it localizes both to the nuclei and cytoplasm in plant cells. MiSSP7.6 protein was shown to interact with two Populus Trihelix transcription factors. Furthermore, when coexpressed with one of the Trihelix transcription factors, MiSSP7.6 is localized to plant nuclei only. Our data suggest that MiSSP7.6 is a novel secreted symbiotic effector and is a potential determinant for ectomycorrhiza formation.

Conservation and expansion of a necrosis-inducing small secreted protein family from host-variable phytopathogens of the Sclerotiniaceae.

Fungal effector proteins facilitate host‐plant colonization and have generally been characterized as small secreted proteins (SSPs). We classified and functionally tested SSPs from the secretomes of three closely related necrotrophic phytopathogens: Ciborinia camelliae, Botrytis cinerea, and Sclerotinia sclerotiorum. Alignment of predicted SSPs identified a large protein family that share greater than 41% amino acid identity and that have key characteristics of previously described microbe‐associated molecular patterns (MAMPs). Strikingly, 73 of the 75 SSP family members were predicted within the secretome of the host‐specialist C. camelliae with single‐copy homologs identified in the secretomes of the host generalists S. sclerotiorum and B. cinerea. To explore the potential function of this family of SSPs, 10 of the 73 C. camelliae proteins, together with the single‐copy homologs from S. sclerotiorum (SsSSP3) and B. cinerea (BcSSP2), were cloned and expressed as recombinant proteins. Infiltration of SsSSP3 and BcSSP2 into host tissue induced rapid necrosis. In contrast, only one of the 10 tested C. camelliae SSPs was able to induce a limited amount of necrosis. Analysis of chimeric proteins consisting of domains from both a necrosis‐inducing and a non‐necrosis‐inducing SSP demonstrated that the C‐terminus of the S. sclerotiorum SSP is essential for necrosis‐inducing function. Deletion of the BcSSP2 homolog from B. cinerea did not affect growth or pathogenesis. Thus, this research uncovered a family of highly conserved SSPs present in diverse ascomycetes that exhibit contrasting necrosis‐inducing functions.

Manipulating the Expression of Small Secreted Protein 1 (Ssp1) Alters Patterns of Development and Metabolism in the White-Rot Fungus Pleurotus ostreatus.

The function of small secreted proteins (SSPs) in saprotrophic fungi is, for the most part, unknown. The white-rot mushroom Pleurotus ostreatus produces considerable amounts of SSPs at the onset of secondary metabolism, during colony development, and in response to chemical compounds such as 5-hydroxymethylfurfural and aryl alcohols. Genetic manipulation of Ssp1, by knockdown (KDssp1) or overexpression (OEssp1), indicated that they are, in fact, involved in the regulation of the ligninolytic system. To elucidate their potential involvement in fungal development, quantitative secretome analysis was performed during the trophophase and the idiophase and at a transition point between the two growth phases. The mutations conferred a time shift in the secretion and expression patterns: OEssp1 preceded the entrance to idiophase and secondary metabolism, while KDssp1 was delayed. This was also correlated with expression patterns of selected genes. The KDssp1 colony aged at a slower pace, accompanied by a slower decline in biomass over time. In contrast, the OEssp1 strain exhibited severe lysis and aging of the colony at the same time point. These phenomena were accompanied by variations in yellow pigment production, characteristic of entrance of the wild type into idiophase. The pigment was produced earlier and in a larger amount in the OEssp1 strain and was absent from the KDssp1 strain. Furthermore, the dikaryon harboring OEssp1 exhibited a delay in the initiation of fruiting body formation as well as earlier aging. We propose that Ssp1 might function as a part of the fungal communication network and regulate the pattern of fungal development and metabolism in P. ostreatusIMPORTANCE Small secreted proteins (SSPs) are common in fungal saprotrophs, but their roles remain elusive. As such, they comprise part of a gene pool which may be involved in governing fungal lifestyles not limited to symbiosis and pathogenicity, in which they are commonly referred to as "effectors." We propose that Ssp1 in the white-rot fungus Pleurotus ostreatus regulates the transition from primary to secondary metabolism, development, aging, and fruiting body initiation. Our observations uncover a novel regulatory role of effector-like SSPs in a saprotroph, suggesting that they may act in fungal communication as well as in response to environmental cues. The presence of Ssp1 homologues in other fungal species supports a common potential role in environmental sensing and fungal development.

Laccaria bicolor MiSSP8 is a small‐secreted protein decisive for the establishment of the ectomycorrhizal symbiosis

The ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems sustaining tree growth and health. Its establishment and functioning implies a long-term and intimate relationship between the soil-borne fungi and the roots of trees. Mycorrhiza-induced Small-Secreted Proteins (MiSSPs) are hypothesized as keystone symbiotic proteins, required to set up the symbiosis by modifying the host metabolism and/or building the symbiotic interfaces. L. bicolor MiSSP8 is the third most highly induced MiSSPs in symbiotic tissues and it is also expressed in fruiting bodies. The MiSSP8-RNAi knockdown mutants are strongly impaired in their mycorrhization ability with Populus, with the lack of fungal mantle and Hartig net development due to the lack of hyphal aggregation. MiSSP8 C-terminus displays a repetitive motif containing a kexin cleavage site, recognized by KEX2 in vitro. This suggests MiSSP8 protein might be cleaved into small peptides. Moreover, the MiSSP8 repetitive motif is found in other proteins predicted secreted by both saprotrophic and ectomycorrhizal fungi. Thus, our data indicate that MiSSP8 is a small-secreted protein involved at early stages of ectomycorrhizal symbiosis, likely by regulating hyphal aggregation and pseudoparenchyma formation.

Chlorophyll fluorescence imaging for monitoring effects of Heterobasidion parviporum small secreted protein induced cell death and in planta defense gene expression

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

Genomics analysis of Aphanomyces spp. identifies a new class of oomycete effector associated with host adaptation

BackgroundOomycetes are a group of filamentous eukaryotic microorganisms that have colonized all terrestrial and oceanic ecosystems, and they include prominent plant pathogens. The Aphanomyces genus is unique in its ability to infect both plant and animal species, and as such exemplifies oomycete versatility in adapting to different hosts and environments. Dissecting the underpinnings of oomycete diversity provides insights into their specificity and pathogenic mechanisms.ResultsBy carrying out genomic analyses of the plant pathogen A. euteiches and the crustacean pathogen A. astaci, we show that host specialization is correlated with specialized secretomes that are adapted to the deconstruction of the plant cell wall in A. euteiches and protein degradation in A. astaci. The A. euteiches genome is characterized by a large repertoire of small secreted protein (SSP)-encoding genes that are highly induced during plant infection, and are not detected in other oomycetes. Functional analysis revealed an SSP from A. euteiches containing a predicted nuclear-localization signal which shuttles to the plant nucleus and increases plant susceptibility to infection.ConclusionCollectively, our results show that Aphanomyces host adaptation is associated with evolution of specialized secretomes and identify SSPs as a new class of putative oomycete effectors.

Specific Hypersensitive Response-Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato.

Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent. C. fulvum strains capable of overcoming one or more of all cloned Cf genes have now emerged. To combat these strains, new Cf genes are required. An effectoromics approach was employed to identify wild tomato accessions carrying new Cf genes. Proteomics and transcriptome sequencing were first used to identify 70 apoplastic in planta-induced C. fulvum SSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe-microbe interactions in planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs, using the Potato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry new Cf genes available for incorporation into cultivated tomato.

A comparative genomic analysis of putative pathogenicity genes in the host-specific sibling species Colletotrichum graminicola and Colletotrichum sublineola

BackgroundColletotrichum graminicola and C. sublineola cause anthracnose leaf and stalk diseases of maize and sorghum, respectively. In spite of their close evolutionary relationship, the two species are completely host-specific. Host specificity is often attributed to pathogen virulence factors, including specialized secondary metabolites (SSM), and small-secreted protein (SSP) effectors. Genes relevant to these categories were manually annotated in two co-occurring, contemporaneous strains of C. graminicola and C. sublineola. A comparative genomic and phylogenetic analysis was performed to address the evolutionary relationships among these and other divergent gene families in the two strains.ResultsInoculation of maize with C. sublineola, or of sorghum with C. graminicola, resulted in rapid plant cell death at, or just after, the point of penetration. The two fungal genomes were very similar. More than 50% of the assemblies could be directly aligned, and more than 80% of the gene models were syntenous. More than 90% of the predicted proteins had orthologs in both species. Genes lacking orthologs in the other species (non-conserved genes) included many predicted to encode SSM-associated proteins and SSPs. Other common groups of non-conserved proteins included transporters, transcription factors, and CAZymes. Only 32 SSP genes appeared to be specific to C. graminicola, and 21 to C. sublineola. None of the SSM-associated genes were lineage-specific. Two different strains of C. graminicola, and three strains of C. sublineola, differed in no more than 1% percent of gene sequences from one another.ConclusionsEfficient non-host recognition of C. sublineola by maize, and of C. graminicola by sorghum, was observed in epidermal cells as a rapid deployment of visible resistance responses and plant cell death. Numerous non-conserved SSP and SSM-associated predicted proteins that could play a role in this non-host recognition were identified. Additional categories of genes that were also highly divergent suggested an important role for co-evolutionary adaptation to specific host environmental factors, in addition to aspects of initial recognition, in host specificity. This work provides a foundation for future functional studies aimed at clarifying the roles of these proteins, and the possibility of manipulating them to improve management of these two economically important diseases.

Impact of biotic and abiotic factors on the expression of fungal effector-encoding genes in axenic growth conditions

In phytopathogenic fungi, the expression of hundreds of small secreted protein (SSP)-encoding genes is induced upon primary infection of plants while no or a low level of expression is observed during vegetative growth. In some species such as Leptosphaeria maculans, this coordinated in-planta upregulation of SSP-encoding genes expression relies on an epigenetic control but the signals triggering gene expression in-planta are unknown. In the present study, biotic and abiotic factors that may relieve suppression of SSP-encoding gene expression during axenic growth of L. maculans were investigated. Some abiotic factors (temperature, pH) could have a limited effect on SSP gene expression. In contrast, two types of cellular stresses induced by antibiotics (cycloheximide, phleomycin) activated strongly the transcription of SSP genes. A transcriptomic analysis to cycloheximide exposure revealed that biological processes such as ribosome biosynthesis and rRNA processing were induced whereas important metabolic pathways such as glycogen and nitrogen metabolism, glycolysis and tricarboxylic acid cycle activity were down-regulated. A quantitatively different expression of SSP-encoding genes compared to plant infection was also detected. Interestingly, the same physico-chemical parameters as those identified here for L. maculans effectors were identified to regulate positively or negatively the expression of bacterial effectors. This suggests that apoplastic phytopathogens may react to similar physiological parameters for regulation of their effector genes.

HIC1 Silencing in Triple-Negative Breast Cancer Drives Progression through Misregulation of LCN2

The tumor suppressor gene HIC1 is frequently deleted or epigenetically silenced in human cancer, where its restoration may improve cancer prognosis. Here, we report results illuminating how HIC1 silencing alters effect or signals in triple-negative breast cancer (TNBC), which are crucial for its pathogenesis. HIC1 expression was silenced only in TNBC compared with other molecular subtypes of breast cancer. Restoring HIC1 expression in TNBC cells reduced cell migration, invasion, and metastasis, whereas RNAi-mediated silencing of HIC1 in untransformed human breast cells increased their invasive capabilities. Mechanistic investigations identified the small-secreted protein lipocalin-2 (LCN2), as a critical downstream target of HIC1 in TNBC cells. Elevating LCN2 expression in cells expressing HIC1 partially rescued its suppression of cell invasion and metastasis. Notably, autocrine secretion of LCN2 induced by loss of HIC1 activated the AKT pathway through the neutrophil gelatinase-associated lipocalin receptor, which is associated with TNBC progression. Taken together, our findings revealed that the HIC1-LCN2 axis may serve as a subtype-specific prognostic biomarker, providing an appealing candidate target for TNBC therapy.

Comparative genome structure, secondary metabolite, and effector coding capacity across Cochliobolus pathogens.

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP–encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.

Characterization of a cassiicolin-encoding gene from Corynespora cassiicola, pathogen of rubber tree (Hevea brasiliensis)

Corynespora Leaf Fall (CLF) is a major disease of rubber tree (Hevea brasiliensis) caused by the Ascomycota Corynespora cassiicola. Here we describe the cloning and characterization of a gene encoding cassiicolin (Cas), a glycosylated cystein-rich small secreted protein (SSP) identified as a potential CLF disease effector in rubber tree. Three isolates with contrasted levels of aggressiveness were analyzed comparatively. The cassiicolin gene was detected – and the toxin successfully purified – from the isolates with high and medium aggressiveness (CCP and CCAM3 respectively) but not from the isolate with the lowest aggressiveness (CCAM1), suggesting the existence of a different disease effector in the later. CCP and CCAM3 carried strictly identical cassiicolin genes and produced toxins of identical mass, as evidence by mass spectrometry analysis, thus suggesting conserved post-translational modifications in addition to sequence identity. The differences in aggressiveness between CCP and CCAM3 may be attributed to differences in cassiicolin transcript levels rather than qualitative variations in cassiicolin structure. Cassiicolin may play an important role in the early phase of infection since a peak of cassiicolin transcripts occurred in 1 or 2 days after inoculation (before the occurrence of the first symptoms), in both the tolerant and the susceptible cultivars.

A Secreted Effector Protein of Laccaria bicolor Is Required for Symbiosis Development

Soil-borne mutualistic fungi, such as the ectomycorrhizal fungi, have helped shape forest communities worldwide over the last 180 million years through a mutualistic relationship with tree roots in which the fungal partner provides a large array of nutrients to the plant host in return for photosynthetically derived sugars. This exchange is essential for continued growth and productivity of forest trees, especially in nutrient-poor soils. To date, the signals from the two partners that mediate this symbiosis have remained uncharacterized. Here we demonstrate that MYCORRHIZAL iNDUCED SMALL SECRETED PROTEIN 7 (MiSSP7), the most highly symbiosis-upregulated gene from the ectomycorrhizal fungus Laccaria bicolor, encodes an effector protein indispensible for the establishment of mutualism. MiSSP7 is secreted by the fungus upon receipt of diffusible signals from plant roots, imported into the plant cell via phosphatidylinositol 3-phosphate-mediated endocytosis, and targeted to the plant nucleus where it alters the transcriptome of the plant cell. L. bicolor transformants with reduced expression of MiSSP7 do not enter into symbiosis with poplar roots. MiSSP7 resembles effectors of pathogenic fungi, nematodes, and bacteria that are similarly targeted to the plant nucleus to promote colonization of the plant tissues and thus can be considered a mutualism effector.