Putative Secretory Proteins Research Articles

A Comprehensive Transcriptomic and Proteomics Analysis of Candidate Secretory Proteins in Rose Grain Aphid, Metopolophium dirhodum (Walker)

The Rose grain aphid, a notable agricultural pest, releases saliva while feeding. Yet, there is a need for a comprehensive understanding of the specific identity and role of secretory proteins released during probing and feeding. Therefore, a combined transcriptomic and proteomic approach was employed in this study to identify putative secretory proteins. The transcriptomic sequencing result led to the assembly of 18,030 unigenes out of 31,344 transcripts. Among these, 705 potential secretory proteins were predicted and functionally annotated against publicly accessible protein databases. Notably, a substantial proportion of secretory genes (71.5%, 69.08%, and 60.85%) were predicted to encode known proteins in Nr, Pfam, and Swiss-Prot databases, respectively. Conversely, 27.37% and 0.99% of gene transcripts were predicted to encode known proteins with unspecified functions in the Nr and Swiss-Prot databases, respectively. Meanwhile, the proteomic analysis result identified, 15 salivary proteins. Interestingly, most salivary proteins (i.e., 60% of the proteins) showed close similarity to A. craccivora, while 46.67% showed close similarity to A. glycines, M. sacchari and S. flava. However, to verify the expression of these secretory genes and characterize the biological function of salivary proteins further investigation should be geared towards gene expression and functional analysis.

Proteomic Analysis of Salivary Secretions from the Tea Green Leafhopper, Empoasca flavescens Fabrecius.

Saliva plays a crucial role in shaping the compatibility of piercing-sucking insects with their host plants. Understanding the complex composition of leafhopper saliva is important for developing effective and eco-friendly control strategies for the tea green leafhopper, Empoasca flavescens Fabrecius, a major piercing-sucking pest in Chinese tea plantations. This study explored the saliva proteins of tea green leafhopper adults using a custom collection device, consisting of two layers of Parafilm stretched over a sucrose diet. A total of 152 proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following the filter-aided sample preparation (FASP). These proteins were categorized into six groups based on their functions, including enzymes, transport proteins, regulatory proteins, cell structure proteins, other proteins, and unknown proteins. Bioinformatics analyses predicted 16 secreted proteins, which were successfully cloned and transcriptionally analyzed across various tissues and developmental stages. Genes encoding putative salivary secretory proteins, including Efmucin1, EfOBP1, EfOBP2, EfOBP3, Efmucin2, low-density lipoprotein receptor-related protein (EfLRP), EFVg1, and EFVg2, exhibited high expressions in salivary gland (SG) tissues and feeding-associated expressions at different developmental stages. These findings shed light on the potential elicitors or effectors mediating the leafhopper feeding and defense responses in tea plants, providing insights into the coevolution of tea plants and leafhoppers. The study's conclusions open avenues for the development of innovative leafhopper control technologies that reduce the reliance on pesticides in the tea industry.

Comparative genomics analysis reveals sequence characteristics potentially related to host preference in Cryptosporidium xiaoi

Cryptosporidium spp. are important diarrhea-associated pathogens in humans and livestock. Among the known species, Cryptosporidium xiaoi, which causes cryptosporidiosis in sheep and goats, was previously recognized as a genotype of the bovine-specific Cryptosporidium bovis based on their high sequence identity in the ssrRNA gene. However, the lack of genomic data has limited characterization of the genetic differences between the two closely related species. In this study, we sequenced the genomes of two C. xiaoi isolates and performed comparative genomic analysis to identify the sequence uniqueness of this ovine-adapted species compared with other Cryptosporidium spp. Our results showed that C. xiaoi is genetically related to C. bovis as shown by their 95.8% genomic identity and similar gene content. Consistent with this, both C. xiaoi and C. bovis appear to have fewer genes encoding mitochondrial metabolic enzymes and invasion-related protein families. However, they appear to possess several species-specific genes. Further analysis indicates that the sequence differences between these two Cryptosporidium spp. are mainly in 24 highly polymorphic genes, half of which are located in the subtelomeric regions. Some of these subtelomeric genes encode secretory proteins that have undergone positive selection. In addition, the genomes of two C. xiaoi isolates, identified as subtypes XXIIIf and XXIIIh, share 99.9% nucleotide sequence identity, with six highly divergent genes encoding putative secretory proteins. Therefore, these species-specific genes and sequence polymorphism in subtelomeric genes probably contribute to the different host preference of C. xiaoi and C. bovis.

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

Complete genome analysis of pathogenic Metschnikowia bicuspidata strain MQ2101 isolated from diseased ridgetail white prawn, Exopalaemon carinicauda

BackgroundMetschnikowia bicuspidata is a pathogenic yesst that can cause disease in many different economic aquatic animal species. In recent years, there was a new disease outbreak in ridgetail white prawn (Exopalaemon carinicauda) in coastal areas of Jiangsu Province China that was referred to as zombie disease by local farmers. The pathogen was first isolated and identified as M. bicuspidata. Although the pathogenicity and pathogenesis of this pathogen in other animals have been reported in some previous studies, research on its molecular mechanisms is still very limited. Therefore, a genome-wide study is necessary to better understand the physiological and pathogenic mechanisms of M. bicuspidata.ResultIn this study, we obtained a pathogenic strain, MQ2101, of M. bicuspidata from diseased E. carinicauda and sequenced its whole genome. The size of the whole genome was 15.98 Mb, and it was assembled into 5 scaffolds. The genome contained 3934 coding genes, among which 3899 genes with biological functions were annotated in multiple underlying databases. In KOG database, 2627 genes were annotated, which were categorized into 25 classes including general function prediction only, posttranslational modification, protein turnover, chaperones, and signal transduction mechanisms. In KEGG database, 2493 genes were annotated, which were categorized into five classes, including cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems. In GO database, 2893 genes were annotated, which were mainly classified in cell, cell part, cellular processes and metabolic processes. There were 1055 genes annotated in the PHI database, accounting for 26.81% of the total genome, among which 5 genes were directly related to pathogenicity (identity ≥ 50%), including hsp90, PacC, and PHO84. There were also some genes related to the activity of the yeast itself that could be targeted by antiyeast drugs. Analysis based on the DFVF database showed that strain MQ2101 contained 235 potential virulence genes. BLAST searches in the CAZy database showed that strain MQ2101 may have a more complex carbohydrate metabolism system than other yeasts of the same family. In addition, two gene clusters and 168 putative secretory proteins were predicted in strain MQ2101, and functional analysis showed that some of the secretory proteins may be directly involved in the pathogenesis of the strain. Gene family analysis with five other yeasts revealed that strain MQ2101 has 245 unique gene families, including 274 genes involved in pathogenicity that could serve as potential targets.ConclusionGenome-wide analysis elucidated the pathogenicity-associated genes of M. bicuspidate while also revealing a complex metabolic mechanism and providing putative targets of action for the development of antiyeast drugs for this pathogen. The obtained whole-genome sequencing data provide an important theoretical basis for transcriptomic, proteomic and metabolic studies of M. bicuspidata and lay a foundation for defining its specific mechanism of host infestation.

In Silico Characterization of the Secretome of the Fungal Pathogen Thielaviopsis punctulata, the Causal Agent of Date Palm Black Scorch Disease

The black scorch disease of date palm caused by Thielaviopsis punctulata is a serious threat to the cultivation and productivity of date palm in Arabian Peninsula. The virulence factors that contribute to pathogenicity of T. punctulata have not been identified yet. In the present study, using bioinformatics approach, secretory proteins of T. punctulata were identified and functionally characterized. A total of 197 putative secretory proteins were identified, of which 74 were identified as enzymes for carbohydrate degradation (CAZymes), 25 were proteases, and 47 were predicted as putative effectors. Within the CAZymes, 50 cell wall-degrading enzymes, potentially to degrade cell wall components such as cellulose, hemicellulose, lignin, and pectin, were identified. Of the 47 putative effectors, 34 possessed at least one functional domain. The secretome of T. punctulata was compared to the predicted secretome of five closely related species (T. musarum, T. ethacetica, T. euricoi, T. cerberus, and T. populi) and identified species specific CAZymes and putative effector genes in T. punctulata, providing a valuable resource for the research aimed at understanding the molecular mechanism underlying the pathogenicity of T. punctulata on Date palm.

Genome-Wide Analysis and Characterization of the Riemerella anatipestifer Putative T9SS Secretory Proteins with a Conserved C-Terminal Domain.

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Recent studies have shown that the R. anatipestifer type IX secretion system (T9SS) acts as a crucial virulence factor. We previously identified two T9SS component proteins, GldK and GldM, and one T9SS effector metallophosphoesterase, which play important roles in bacterial virulence. In this study, 19 T9SS-secreted proteins that contained a conserved T9SS C-terminal domain (CTD) were predicted in R. anatipestifer strain Yb2 by searching for CTD-encoding sequences in the whole genome. The proteins were confirmed with a liquid chromatography-tandem mass spectrometry analysis of the bacterial culture supernatant. Nine of them were reported in our previous study. We generated recombinant proteins and mouse antisera for the 19 predicted proteins to confirm their expression in the bacterial culture supernatant and in bacterial cells. Western blotting indicated that the levels of 14 proteins were significantly reduced in the T9SS mutant Yb2ΔgldM culture medium but were increased in the bacterial cells. RT-qPCR indicated that the expression of these genes did not differ between the wild-type strain Yb2 and the T9SS mutant Yb2ΔgldM. Nineteen mutant strains were successfully constructed to determine their virulence and proteolytic activity, which indicated that seven proteins are associated with bacterial virulence, and two proteins, AS87_RS04190 and AS87_RS07295, are protease-activity-associated virulence factors. In summary, we have identified at least 19 genes encoding T9SS-secreted proteins in the R. anatipestifer strain Yb2 genome, which encode multiple functions associated with the bacterium's virulence and proteolytic activity. IMPORTANCE Riemerella anatipestifer T9SS plays an important role in bacterial virulence. We have previously reported nine R. anatipestifer T9SS-secreted proteins and clarified the function of the metallophosphoesterase. In this study, we identified 10 more secreted proteins associated with the R. anatipestifer T9SS, in addition to the nine previously reported. Of these, 14 proteins showed significantly reduced secretion into the bacterial culture medium but increased expression in the bacterial cells of the T9SS mutant Yb2ΔgldM; seven proteins were shown to be associated with bacterial virulence; and two proteins, AS87_RS04190 and AS87_RS07295, were shown to be protease-activity-associated virulence factors. Thus, we have demonstrated that multiple R. anatipestifer T9SS-secreted proteins function in virulence and proteolytic activity.

Proteomic Analysis Reveals That Placenta-Specific Protein 9 Inhibits Proliferation and Stimulates Motility of Human Bronchial Epithelial Cells.

Placenta-specific protein 9 (PLAC9) is a putative secretory protein that was initially identified in the placenta and is involved in cell proliferation and motility. Bioinformatics analyses revealed that PLAC9 is repressed in lung cancers (LCs), especially lung adenocarcinomas, compared to that in the paired adjacent normal tissues, indicating that PLAC9 might be involved in the pathogenesis of pulmonary diseases. To investigate the potential role of PLAC9 in the abnormal reprogramming of airway epithelial cells (AECs), a key cause of pulmonary diseases, we constructed a stable PLAC9-overexpressing human bronchial epithelial cell line (16HBE-GFP-Plac9). We utilized the proteomic approach isobaric tag for relative and absolute quantification (iTRAQ) to analyze the effect of PLAC9 on cellular protein composition. Gene ontology (GO) and pathway analyses revealed that GO terms and pathways associated with cell proliferation, cell cycle progression, and cell motility and migration were significantly enriched among the proteins regulated by PLAC9. Our in vitro results showed that PLAC9 overexpression reduced cell proliferation, altered cell cycle progression, and increased cell motility, including migration and invasion. Our findings suggest that PLAC9 inhibits cell proliferation through S phase arrest by altering the expression levels of cyclin/cyclin-dependent kinases (CDKs) and promotes cell motility, likely via the concerted actions of cyclins, E-cadherin, and vimentin. Since these mechanisms may underlie PLAC9-mediated abnormal human bronchial pathogenesis, our study provides a basis for the development of molecular targeted treatments for LCs.

Whole-Genome Sequence Resource of Calonectria ilicicola, the Casual Pathogen of Soybean Red Crown Rot

Calonectria ilicicola (ana. Cylindrocladium parasiticum) is a soilborne plant pathogenic fungus with a broad host range, and it can cause red crown rot of soybean and Cylindrocladium black rot of peanut, which has become an emerging threat to crop production worldwide. Limited molecular studies have focused on Calonectria ilicicola and one of the possible difficulties is the lack of genomic resource. This study presents the first high quality and near-completed genome of C. ilicicola using the Oxford Nanopore GridION sequencing platform. A total of 16 contigs were assembled and the genome of C. ilicicola isolate F018 was estimated to have 11 chromosomes. Currently, the C. ilicicola F018 genome represents the most contiguous assembly, which has the lowest contig number and the highest contig N50 among all Calonectria genome resources. Putative protein-coding sequences and secretory proteins were estimated to be 17,308 and 1,930 in the C. ilicicola F018 genome, respectively; and the prediction was close to other plant pathogenic fungi such as Fusarium species within the Nectriaceae family. The availability of this high-quality genome resource is expected to facilitate research on fungal biology and genetics of C. ilicicola, and to support the understanding on pathogen virulence and disease management.

Transcriptomic changes in the pre-parasitic juveniles of Meloidogyne incognita induced by silencing of effectors Mi-msp-1 and Mi-msp-20.

Plant-parasitic root-knot nematode Meloidogyne incognita uses an array of effector proteins to establish successful plant infections. Mi-msp-1 and Mi-msp-20 are two known effectors secreted from nematodesubventral oesophageal glands; Mi-msp-1 being a putative secretory venom allergen AG5-like protein, whereas Mi-msp-20 isa pioneer gene with a coiled-coil motif. Expression of specific effector is known to cause disturbances in the expression of other effectors. Here, we used RNA-Seq to investigate the pleiotropic effects of silencing Mi-msp-1 and Mi-msp-20. A total of 25.1-51.9 million HQ reads generated from Mi-msp-1 and Mi-msp-20 silenced second-stage juveniles (J2s) along with freshly hatched J2s were mapped to an already annotated M. incognita proteome to understand the impact on various nematode pathways. As compared to control, silencing of Mi-msp-1 caused differential expression of 29 transcripts, while Mi-msp-20 silencing resulted in differential expression of a broader set of 409 transcripts. In the Mi-msp-1 silenced J2s, cytoplasm (GO:0005737) was the most enriched gene ontology (GO) term, whereas in the Mi-msp-20 silenced worms, embryo development (GO:0009792), reproduction (GO:0000003) and nematode larval development (GO:0002119) were the most enriched terms. Limited crosstalk was observed between these two effectors as a sheer 5.9% of the up-regulated transcripts were common between Mi-msp-1 and Mi-msp-20 silenced nematodes. Our results suggest that in addition to the direct knock-down caused by silencing of Mi-msp-1 and Mi-msp-20, the cascading effect on other genes might also be contributing to a reduction in nematode's parasitic abilities.

Comparative secretome analysis of Indian wheat leaf rust pathogen Puccinia triticina

The secretome of two races, 77-5 and 106 of wheat leaf rust pathogen Puccinia triticina with known virulence and avirulent trait, respectively were analyzed in this study. The secretome analysis revealed 546 putative secretory proteins (PSPs) present in the race 77-5, and 481 PSPs in race 106. Race-specific PSPs analysis also showed that race 77-5 had higher number of PSPs (19.72%) in comparison to race 106. Various other gene families like pathogenicity and virulence factor were also expanded in higher number in the race 77-5 and few of them having multiple domains which are known for pathogenicity, were exclusively present in this virulent race. The candidate secretory effector proteins (CSEP) analysis also showed that the virulent race contained 8.98% higher CSEP proteins compared to the avirulent race 106. The results suggest that these genes are playing important roles in their respective race-specific manner. Surprisingly the carbohydrate metabolism-related enzymes were found 5.26% higher in the avirulent race in comparison to the virulent one, and few of them have shown race specificity. The in-silico expression analysis of the selected candidate's genes also revealed their role in the pathogenesis process. The three dimensional (3D) structure predictions were performed for few of the candidate genes that were highly expressed. In this comparative secretome analysis, our findings provide a baseline for the characterization of effectors and avirulence factors in different races of P. triticina.

Zinc binding proteome of a phytopathogen Xanthomonas translucens pv. undulosa.

Xanthomonas translucens pv. undulosa (Xtu) is a proteobacteria which causes bacterial leaf streak (BLS) or bacterial chaff disease in wheat and barley. The constant competition for zinc (Zn) metal nutrients contributes significantly in plant–pathogen interactions. In this study, we have employed a systematic in silico approach to study the Zn-binding proteins of Xtu. From the whole proteome of Xtu, we have identified approximately 7.9% of proteins having Zn-binding sequence and structural motifs. Further, 115 proteins were found homologous to plant–pathogen interaction database. Among these 115 proteins, 11 were predicted as putative secretory proteins. The functional diversity in Zn-binding proteins was revealed by functional domain, gene ontology and subcellular localization analysis. The roles of Zn-binding proteins were found to be varied in the range from metabolism, proteolysis, protein biosynthesis, transport, cell signalling, protein folding, transcription regulation, DNA repair, response to oxidative stress, RNA processing, antimicrobial resistance, DNA replication and DNA integration. This study provides preliminary information on putative Zn-binding proteins of Xtu which may further help in designing new metal-based antimicrobial agents for controlling BLS and bacterial chaff infections on staple crops.

Placenta-specific 9, a putative secretory protein, induces G2/M arrest and inhibits the proliferation of human embryonic hepatic cells.

Background: Placenta-specific 9 (Plac9) is a putative secreted protein that was first discovered in the context of embryogenesis. The expression pattern of Plac9 during embryogenesis, together with the results of recent reports, suggest that Plac9 may play a role in the liver development. The present study was conducted to investigate the secretory characteristics of Plac9 and its potential role in liver cell physiology. Methods: Immunofluorescence was employed to identify the subcellular distribution of Plac9. Cellular proliferative activity was analyzed by MTT assay and cell colony formation. The cell cycle distribution of Plac9 was analyzed by flow cytometry, and a functional analysis was performed using L02 cells following their stable infection with a lentivirus over-expressing Plac9. Results: Plac9 is a novel protein that is localized to the cytoplasm and may be secreted through the classic endoplasmic reticulum-Golgi route. The overexpression of Plac9 inhibits cell growth and induces G2/M phase arrest. Conclusion: Our findings reveal a novel role for Plac9 in regulating cell growth.

Annotation resource of tandem repeat-containing secretory proteins in sixty fungi

Fungal secretory proteins that interact with host plants are regarded as effectors. Because fungal effectors rarely contain conserved sequence features, identification and annotation of fungal effectors from predicted secretory proteins are difficult using outward comparison methods such as BLAST or hidden Markov model. In desire of more sequence features to prioritize research interests of fungal secretory proteins, this study developed a pipeline to identify tandem repeat (TR) domain within putative secretory proteins and tested a hypothesis that at least one type of TR domain in non-orthologous secretory proteins has emerged from convergent evolution for plant pathogenicity. There were 2804 types of TR domains and a total of 2925 TR-containing secretory proteins found from 60 fungi. There was no conserved type of TR domain shared only by plant pathogens, indicating functional divergence for different types of TR domain and TR-containing secretory proteins. The annotation resource of putative fungal TR-containing secretory proteins provides new sequence features that will be useful for the community interested in fungal effector biology.

Proteomic analysis of lysine acetylation provides strong evidence for involvement of acetylated proteins in plant meiosis and tapetum function.

Protein lysine acetylation (KAC) is a dynamic and reversible post-translational modification that has important biological roles in many organisms. Although KAC has been shown to affect reproductive development and meiosis in yeast and animals, similar studies are largely lacking in flowering plants, especially proteome-scale investigations for particular reproductive stages. Here, we report results from a proteomic investigation to detect the KAC status of the developing rice anthers near the time of meiosis (RAM), providing strong biochemical evidence for roles of many KAC-affected proteins during anther development and meiosis in rice. We identified a total of 1354 KAC sites in 676 proteins. Among these, 421 acetylated proteins with 629 KAC sites are novel, greatly enriching our knowledge on KAC in flowering plants. Gene Ontology enrichment analysis showed chromatin silencing, protein folding, fatty acid biosynthetic process and response to stress to be over-represented. In addition, certain potentially specific KAC motifs in RAM were detected. Importantly, 357 rice meiocyte proteins were acetylated; and four proteins genetically identified to be important for rice tapetum and pollen development were acetylated on 14 KAC sites in total. Furthermore, 47 putative secretory proteins were detected to exhibit acetylated status in RAM. Moreover, by comparing our lysine acetylome with the RAM phosphoproteome we obtained previously, we proposed a correlation between KAC and phosphorylation as a potential modulatory mechanism in rice RAM. This study provides the first global survey of KAC in plant reproductive development, making a promising starting point for further functional analysis of KAC during rice anther development and meiosis.

Transcriptome analysis of the salivary glands of the grain aphid, Sitobion avenae

Aphid saliva plays important roles in aphid-host interactions, such as assisting aphid digestion, detoxification, activating or suppressing plant defenses. The grain aphid, Sitobion avenae, is one of the most devastating pests of cereals worldwide. In this study, we performed the transcriptome analysis of salivary glands of S. avenae. A total of 33,079 assembled unigenes were identified in the salivary glands of aphids. Of the all obtained unigenes, 15,833(47.86%) and 10,829(32.73%) unigenes showed high similarity to known proteins in Nr and Swiss-Prot databases respectively. 526 unigenes were predicted to encode secretory proteins, including some digestive and detoxifying enzymes and potential effectors. The RT-PCR and RT-qPCR results showed that all of the 15 most highly expressed putative secretory proteins specifically expressed in salivary glands. Interestingly, 11 of the 15 most highly expressed putative secretory proteins were still not matched to function-known proteins. We also detected the expression of 9 interested putative secretory proteins in aphid different tissues, including some digestive and detoxifying enzymes, effectors and Ca2+ binding proteins. The results showed that only glutathione-S-transferase 1 was specifically expressed in salivary glands. These findings provide a further insight into the identification of potential effectors involving in aphid-cereals interactions.

Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding

BackgroundTicks secrete a diverse mixture of secretory proteins into the host to evade its immune response and facilitate blood-feeding, making secretory proteins attractive targets for the production of recombinant anti-tick vaccines. The largely neglected tick species, Rhipicephalus zambeziensis, is an efficient vector of Theileria parva in southern Africa but its available sequence information is limited. Next generation sequencing has advanced sequence availability for ticks in recent years and has assisted the characterisation of secretory proteins. This study focused on the de novo assembly and annotation of the salivary gland transcriptome of R. zambeziensis and the temporal expression of secretory protein transcripts in female and male ticks, before the onset of feeding and during early and late feeding.ResultsThe sialotranscriptome of R. zambeziensis yielded 23,631 transcripts from which 13,584 non-redundant proteins were predicted. Eighty-six percent of these contained a predicted start and stop codon and were estimated to be putatively full-length proteins. A fifth (2569) of the predicted proteins were annotated as putative secretory proteins and explained 52% of the expression in the transcriptome. Expression analyses revealed that 2832 transcripts were differentially expressed among feeding time points and 1209 between the tick sexes. The expression analyses further indicated that 57% of the annotated secretory protein transcripts were differentially expressed. Dynamic expression profiles of secretory protein transcripts were observed during feeding of female ticks. Whereby a number of transcripts were upregulated during early feeding, presumably for feeding site establishment and then during late feeding, 52% of these were downregulated, indicating that transcripts were required at specific feeding stages. This suggested that secretory proteins are under stringent transcriptional regulation that fine-tunes their expression in salivary glands during feeding. No open reading frames were predicted for 7947 transcripts. This class represented 17% of the differentially expressed transcripts, suggesting a potential transcriptional regulatory function of long non-coding RNA in tick blood-feeding.ConclusionsThe assembled sialotranscriptome greatly expands the sequence availability of R. zambeziensis, assists in our understanding of the transcription of secretory proteins during blood-feeding and will be a valuable resource for future vaccine candidate selection.

Discovery of a new intravacuolar protein required for the autophagy, development and virulence of Beauveria bassiana.

High proportions of hypothetical proteins exist in genomic databases of fungi, including putative secretory proteins (PSPs) likely involved in fungal invasion and virulence. Here we characterize one of many PSPs revealed in the previous transcriptome of Beauveria bassiana (a fungal insect pathogen) infecting a global lepidopteran pest and name it vacuole-localized protein 4 (VLP4) because this small, domain-lacking protein (22.96 kDa) was specifically localized in the vacuoles of hyphal cells. Deletion of VLP4 resulted in repression of almost all genes acting in autophagy and central development pathways. Consequently, the deletion mutant formed no autophagosome in hyphal vacuoles and displayed severe defects in aerial conidiation. conidial hydrophobicity to the insect surface, and secretion of cuticle-degrading Pr1 proteases required for normal cuticle infection. Blastospore formation was inhibited in the submerged mutant culture mimic to insect haemolymph, and formation of hyphal bodies in vivo was delayed. The fungal virulence was attenuated in the absence of VLP4. These phenotypic defects were well restored by targeted gene complementation. Our findings unveil a vital role of VLP4 in B. bassiana and call attention to many more PSPs for new insights into the interactions of fungal insect pathogens with insects.

Vital role for cyclophilin B (CypB) in asexual development, dimorphic transition and virulence of Beauveria bassiana

Cyclophilin B (CypB) was previously revealed as one of many putative secretory proteins in the transcriptome of Beauveria bassiana infection to a lepidopteran pest. Here we show a main localization of CypB in hyphal cell walls and septa and its essential role in the in vitro and in vivo asexual cycles of the fungal insect pathogen. Deletion of cypB reduced colony growth by 16–42% on two rich media and 30 scant media with different carbon or nitrogen sources. The deletion mutant suffered a delayed conidiation on a standard medium and a final 47% reduction in conidial yield, accompanied with drastic transcript depression of several key genes required for conidiation and conidial maturation. The mutant conidia required 10h longer to germinate 50% at optimal 25°C than wild-type conidia. Intriguingly, cultivation of the mutant conidia in a trehalose-peptone broth mimic to insect hemolymph resulted in 83% reduction in blastospore yield but only slight decrease in biomass level, indicating severe defects in transition of hyphae to blastospores. LT50 for the deletion mutant against Galleria mellonella larvae through normal cuticle infection was prolonged to 7.4d from a wild-type estimate of 4.7d. During colony growth, additionally, the deletion mutant displayed hypersensitivity to Congo red, menadione, H2O2 and heat shock but increased tolerance to cyclosporine A and rapamycin. All of changes were restored by targeted gene complementation. Altogether, CypB takes part in sustaining normal growth, aerial conidiation, conidial germination, dimorphic transition, stress tolerance and pathogenicity in B. bassiana.

The prediction of a pathogenesis-related secretome of Puccinia helianthi through high-throughput transcriptome analysis

BackgroundMany plant pathogen secretory proteins are known to be elicitors or pathogenic factors,which play an important role in the host-pathogen interaction process. Bioinformatics approaches make possible the large scale prediction and analysis of secretory proteins from the Puccinia helianthi transcriptome. The internet-based software SignalP v4.1, TargetP v1.01, Big-PI predictor, TMHMM v2.0 and ProtComp v9.0 were utilized to predict the signal peptides and the signal peptide-dependent secreted proteins among the 35,286 ORFs of the P. helianthi transcriptome.Results908 ORFs (accounting for 2.6% of the total proteins) were identified as putative secretory proteins containing signal peptides. The length of the majority of proteins ranged from 51 to 300 amino acids (aa), while the signal peptides were from 18 to 20 aa long. Signal peptidase I (SpI) cleavage sites were found in 463 of these putative secretory signal peptides. 55 proteins contained the lipoprotein signal peptide recognition site of signal peptidase II (SpII). Out of 908 secretory proteins, 581 (63.8%) have functions related to signal recognition and transduction, metabolism, transport and catabolism. Additionally, 143 putative secretory proteins were categorized into 27 functional groups based on Gene Ontology terms, including 14 groups in biological process, seven in cellular component, and six in molecular function. Gene ontology analysis of the secretory proteins revealed an enrichment of hydrolase activity. Pathway associations were established for 82 (9.0%) secretory proteins. A number of cell wall degrading enzymes and three homologous proteins specific to Phytophthora sojae effectors were also identified, which may be involved in the pathogenicity of the sunflower rust pathogen.ConclusionsThis investigation proposes a new approach for identifying elicitors and pathogenic factors. The eventual identification and characterization of 908 extracellularly secreted proteins will advance our understanding of the molecular mechanisms of interactions between sunflower and rust pathogen and will enhance our ability to intervene in disease states.