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.     

The infection cushion of Botrytis cinerea: a fungal ‘weapon’ of plant-biomass destruction

The necrotrophic plant-pathogen fungus Botrytis cinerea produces multicellular appressoria dedicated to plant penetration, named infection cushions (IC). A microarray analysis was performed to identify genes upregulated in mature IC. The expression data were validated by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and biochemical assays. 1231 upregulated genes and 79 up-accumulated proteins were identified. The data support the secretion of effectors by IC: phytotoxins, ROS, proteases, cutinases, plant cell wall–degrading enzymes and plant cell death–inducing proteins. Parallel upregulation of sugar transport and sugar catabolism–encoding genes would indicate a role of IC in nutrition. The data also reveal a substantial remodelling of the IC cell wall and suggest a role for melanin and chitosan in IC function. Lastly, mutagenesis of two upregulated genes in IC identified secreted fasciclin-like proteins as actors in the pathogenesis of B. cinerea. These results support the role of IC in plant penetration and also introduce other unexpected functions for this fungal organ, in colonization, necrotrophy and nutrition of the pathogen.     

Secretome analysis reveals effector candidates associated with broad host range necrotrophy in the fungal plant pathogen Sclerotinia sclerotiorum

Background

The white mold fungus Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a remarkably broad host range. The interaction of necrotrophs with their hosts is more complex than initially thought, and still poorly understood.

Results

We combined bioinformatics approaches to determine the repertoire of S. sclerotiorum effector candidates and conducted detailed sequence and expression analyses on selected candidates. We identified 486 S. sclerotiorum secreted protein genes expressed in planta, many of which have no predicted enzymatic activity and may be involved in the interaction between the fungus and its hosts. We focused on those showing (i) protein domains and motifs found in known fungal effectors, (ii) signatures of positive selection, (iii) recent gene duplication, or (iv) being S. sclerotiorum-specific. We identified 78 effector candidates based on these properties. We analyzed the expression pattern of 16 representative effector candidate genes on four host plants and revealed diverse expression patterns.

Conclusions

These results reveal diverse predicted functions and expression patterns in the repertoire of S. sclerotiorum effector candidates. They will facilitate the functional analysis of fungal pathogenicity determinants and should prove useful in the search for plant quantitative disease resistance components active against the white mold.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-336) contains supplementary material, which is available to authorized users.     

Calcium Oxalate Crystals: An Integral Component of the Sclerotinia sclerotiorum/Brassica carinata Pathosystem

Oxalic acid is an important virulence factor for disease caused by the fungal necrotrophic pathogen Sclerotinia sclerotiorum, yet calcium oxalate (CaOx) crystals have not been widely reported. B. carinata stems were infected with S. sclerotiorum and observed using light microscopy. Six hours post inoculation (hpi), CaOx crystals were evident on 46% of stem sections and by 72 hpi on 100%, demonstrating that the secretion of oxalic acid by S. sclerotiorum commences before hyphal penetration. This is the first time CaOx crystals have been reported on B. carinata infected with S. sclerotiorum. The shape of crystals varied as infection progressed. Long tetragonal rods were dominant 12 hpi (68% of crystal-containing samples), but by 72 hpi, 50% of stems displayed bipyramidal crystals, and only 23% had long rods. Scanning electron microscopy from 24 hpi revealed CaOx crystals in all samples, ranging from tiny irregular crystals (< 0.5 μm) to large (up to 40 μm) highly organized arrangements. Crystal morphology encompassed various forms, including tetragonal prisms, oval plates, crystal sand, and druses. Large conglomerates of CaOx crystals were observed in the hyphal mass 72 hpi and these are proposed as a strategy of the fungus to hold and detoxify Ca²⁺ions. The range of crystal morphologies suggests that S. sclerotiorum growth and infection controls the form taken by CaOx crystals.     

Secretomes of medically important fungi reflect morphological and phylogenetic diversity

Secretome represents a main target for understanding the mechanisms of fungal adaptation. In the present study, we focus on the secretomes of fungi associated with infections in humans and other mammals in order to explore relationships between the diverse morphological and phylogenetic groups. Almost all the mammalian pathogenic fungi analyzed have secretome sizes smaller than 1000 proteins and, secreted proteins comprise between 5% and 10% of the total proteome. As expected, the correlation pattern between the secretome size and the total proteome was similar to that described in previous secretome studies of fungi. With regard to the morphological groups, minimum secretome sizes of less than 250 secreted proteins and low values for the fraction of secreted proteins are shown in mammalian pathogenic fungi with reduced proteomes such as microsporidia, atypical fungi and some species of yeasts and yeast-like fungi (Malassezia). On the other hand, filamentous fungi have significantly more secreted proteins and the highest numbers are present in species of filamentous fungi that also are plant or insect pathogens (Fusarium verticilloides, Fusarium oxysporum and Basidiobolus meristosporus). With respect to phylogeny, there are also variations in secretome size across fungal subphyla: Microsporidia, Taphrinomycotina, Ustilagomycotina and Saccharomycotina contain small secretomes; whereas larger secretomes are found in Agaricomycotina, Pezizomycotina, Mucoromycotina and Entomophthoromycotina. Finally, principal component analysis (PCA) was conducted on the complete secretomes. The PCA results revealed that, in general, secretomes of fungi belonging to the same morphological group or subphyla cluster together. In conclusion, our results point out that in medically important fungi there is a relationship between the secretome and the morphological group or phylogenetic classification.     

Overexpression of Three Glucosinolate Biosynthesis Genes in Brassica napus Identifies Enhanced Resistance to Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are notorious plant pathogenic fungi with an extensive host range including Brassica crops. Glucosinolates (GSLs) are an important group of secondary metabolites characteristic of the Brassicales order, whose degradation products are proving to be increasingly important in plant protection. Enhancing the defense effect of GSL and their associated degradation products is an attractive strategy to strengthen the resistance of plants by transgenic approaches. We generated the lines of Brassica napus with three biosynthesis genes involved in GSL metabolic pathway (BnMAM1, BnCYP83A1 and BnUGT74B1), respectively. We then measured the foliar GSLs of each transgenic lines and inoculated them with S. sclerotiorum and B. cinerea. Compared with the wild type control, over-expressing BnUGT74B1 in B. napus increased the aliphatic and indolic GSL levels by 1.7 and 1.5 folds in leaves respectively; while over-expressing BnMAM1 or BnCYP83A1 resulted in an approximate 1.5-fold higher only in the aliphatic GSL level in leaves. The results of plant inoculation demonstrated that BnUGT74B1-overexpressing lines showed less severe disease symptoms and tissue damage compared with the wild type control, but BnMAM1 or BnCYP83A1-overexpressing lines showed no significant difference in comparison to the controls. These results suggest that the resistance to S. sclerotiorum and B. cinerea in B. napus could be enhanced through tailoring the GSL profiles by transgenic approaches or molecular breeding, which provides useful information to assist plant breeders to design improved breeding strategies.     

Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from Cynanchum komarovii That Confers Fungal Resistance in Arabidopsis

Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species.Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from Cynanchum komarovii that effectively inhibits polygalacturonases from Botrytis cinerea and Rhizoctonia solani. We found the expression of CkPGIP1 to be induced in response to salicylic acid, wounding, and infection with B. cinerea and R. solani. In addition, transgenic overexpression in Arabidopsis enhanced resistance against B. cinerea. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of B. cinerea and R. solani polygalacturonases by 62.7–66.4% and 56.5–60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the B. cinerea polygalacturonase, and with the C-terminus of the polygalacturonase from R. solani. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.     

Role of camalexin, indole glucosinolates, and side chain modification of glucosinolate-derived isothiocyanates in defense of Arabidopsis against Sclerotinia sclerotiorum

Plant secondary metabolites are known to facilitate interactions with a variety of beneficial and detrimental organisms, yet the contribution of specific metabolites to interactions with fungal pathogens is poorly understood. Here we show that, with respect to aliphatic glucosinolate‐derived isothiocyanates, toxicity against the pathogenic ascomycete Sclerotinia sclerotiorum depends on side chain structure. Genes associated with the formation of the secondary metabolites camalexin and glucosinolate were induced in Arabidopsis thaliana leaves challenged with the necrotrophic pathogen S. sclerotiorum. Unlike S. sclerotiorum, the closely related ascomycete Botrytis cinerea was not identified to induce genes associated with aliphatic glucosinolate biosynthesis in pathogen‐challenged leaves. Mutant plant lines deficient in camalexin, indole, or aliphatic glucosinolate biosynthesis were hypersusceptible to S. sclerotiorum, among them the myb28 mutant, which has a regulatory defect resulting in decreased production of long‐chained aliphatic glucosinolates. The antimicrobial activity of aliphatic glucosinolate‐derived isothiocyanates was dependent on side chain elongation and modification, with 8‐methylsulfinyloctyl isothiocyanate being most toxic to S. sclerotiorum. This information is important for microbial associations with cruciferous host plants and for metabolic engineering of pathogen defenses in cruciferous plants that produce short‐chained aliphatic glucosinolates.     

Fungal Endophyte Diversity and Bioactivity in the Indian Medicinal Plant Ocimum sanctum Linn

Endophytic mycopopulation isolated from India’s Queen of herbs Tulsi (Ocimum sanctum) were explored and investigated for their diversity and antiphytopathogenic activity against widespread plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani and Fusarium oxysporum. 90 fungal isolates, representing 17 genera were recovered from 313 disease-free and surface sterilised plant segments (leaf and stem tissues) from three different geographic locations (Delhi, Hyderabad and Mukteshwar) during distinct sampling times in consequent years 2010 and 2011 in India. Fungal endophytes were subjected to molecular identification based on rDNA ITS sequence analysis. Plant pathogens such as F. verticillioides, B. maydis, C. coarctatum, R. bataticola, Hypoxylon sp., Diaporthe phaseolorum, Alternaria tenuissima and A. alternata have occurred as endophyte only during second sampling (second sampling in 2011) in the present study. Bi-plot generated by principal component analysis suggested tissue specificity of certain fungal endophytes. Dendrogram revealed species abundance as a function of mean temperature of the location at the time of sampling. Shannon diversity in the first collection is highest in Hyderabad leaf tissues (H'' = 1.907) whereas in second collection it was highest from leaf tissues of Delhi (H'' = 1.846). Mukteshwar (altitude: 7500 feet) reported least isolation rate in second collection. Nearly 23% of the total fungal isolates were considered as potent biocontrol agent. Hexane extract of M. phaseolina recovered from Hyderabad in first collection demonstrated highest activity against S. sclerotiorum with IC₅₀ value of 0.38 mg/ml. Additionally, its components 2H-pyran-2-one, 5,6-dihydro-6-pentyl and palmitic acid, methyl ester as reported by GC-MS Chromatogram upon evaluation for their antiphytopathogenic activity exhibited IC₅₀ value of 1.002 and 0.662 against respectively S. sclerotiorum indicating their significant role in antiphytopathogenic activity of hexane extract. The production of 2H-pyran-2-one, 5,6-dihydro-6-pentyl from M. phaseolina, an endophytic fungus is being reported for the first time.     

Mining nematode protein secretomes to explain lifestyle and host specificity

Parasitic nematodes are highly successful pathogens, inflicting disease on humans, animals and plants. Despite great differences in their life cycles, host preference and transmission modes, these parasites share a common capacity to manipulate their host’s immune system. This is at least partly achieved through the release of excretory/secretory proteins, the most well-characterized component of nematode secretomes, that are comprised of functionally diverse molecules. In this work, we analyzed published protein secretomes of parasitic nematodes to identify common patterns as well as species-specific traits. The 20 selected organisms span 4 nematode clades, including plant pathogens, animal parasites, and the free-living species Caenorhabditis elegans. Transthyretin-like proteins were the only component common to all adult secretomes; many other protein classes overlapped across multiple datasets. The glycolytic enzymes aldolase and enolase were present in all parasitic species, but missing from C. elegans. Secretomes from larval stages showed less overlap between species. Although comparison of secretome composition across species and life-cycle stages is challenged by the use of different methods and depths of sequencing among studies, our workflow enabled the identification of conserved protein families and pinpointed elements that may have evolved as to enable parasitism. This strategy, extended to more secretomes, may be exploited to prioritize therapeutic targets in the future.     

Can plant sugars mediate the effect of nitrogen fertilization on lettuce susceptibility to two necrotrophic pathogens: Botrytis cinerea and Sclerotinia sclerotiorum?

Aims

Nitrogen (N) fertilization is known to modify a plant’s susceptibility to necrotrophic diseases. However, the effect of N nutrition on defence is not well known. It was hypothesized that not only molecules related to the N metabolism but also main sugars could mediate the effect of plant fertilization on its susceptibility to pathogens.

Methods

Two necrotrophic fungi, Botrytis cinerea and Sclerotinia sclerotiorum were inoculated on leaves of lettuce plants grown at five nitrate (NO₃ ^?) fertilization levels, in three independent experiments. Variations in plant composition at the time of inoculation were linked to the size of lesions observed after 5–6 days.

Results

Both diseases were favoured by high NO₃ ^? fertilization. However, the highest disease levels were not found in the same experiment for B. cinerea and S. sclerotiorum. Among the components measured, NO₃ ^? and sucrose (SUC) were positively and negatively correlated to the two diseases in the three experiments, but the relationship between SUC and lesion size was more significant for S. sclerotiorum. Water content, N and total carbon (C) were also significantly correlated to the diseases, but the relationships were less straightforward. The ratios of SUC over total sugars and fructose (FRU) over total sugars fitted, very closely for S. sclerotiorum, a negative and positive exponential relationship respectively with lesion size. Absolute or relative glucose levels were not linked to the diseases.

Conclusions

Plant metabolic modifications induced by variations of N availability conferred the plant variable defence ability, which seemed, at least for S. sclerotiorum, mainly mediated by variations in host SUC and FRU levels. The generalization of these findings to other species would be of interest.     

Secret-AAR: a web server to assess the antigenic density of proteins and homology search against bacterial and parasite secretome proteins

The secretome refers to all the Excreted/Secreted (ES) proteins of a cell, and these are involved in critical biological processes, such as cell-cell communication, and host immune responses. Recently, we introduced the Abundance of Antigenic Aegions (AAR) value to assess the protein antigenic density and to evaluate the antigenic potential of secretomes. Here, to facilitate the AAR calculation, we implemented it as a user-friendly webserver. We extended the webserver capabilities implementing a sequence-based tool for searching homologous proteins across secretomes, including experimental and predicted secretomes of Mycobacterium tuberculosis and Taenia solium. Additionally, twelve secretomes of helminths, five of Mycobacterium and two of Gram-negative bacteria are also available. Our webserver is a useful tool for researchers working on immunoinformatics and reverse vaccinology, aiming at discovering candidate proteins for new vaccines or diagnostic tests, and it can be used to prioritize the experimental analysis of proteins for druggability assays. The Secret-AAR web server is available at http://microbiomics.ibt.unam.mx/tools/aar/.     

The AtrbohD-mediated oxidative burst elicited by oligogalacturonides in Arabidopsis is dispensable for the activation of defense responses effective against Botrytis cinerea

Oligogalacturonides (OGs) are endogenous elicitors of defense responses released after partial degradation of pectin in the plant cell wall. We have previously shown that, in Arabidopsis (Arabidopsis thaliana), OGs induce the expression of PHYTOALEXIN DEFICIENT3 (PAD3) and increase resistance to the necrotrophic fungal pathogen Botrytis cinerea independently of signaling pathways mediated by jasmonate, salicylic acid, and ethylene. Here, we illustrate that the rapid induction of the expression of a variety of genes by OGs is also independent of salicylic acid, ethylene, and jasmonate. OGs elicit a robust extracellular oxidative burst that is generated by the NADPH oxidase AtrbohD. This burst is not required for the expression of OG-responsive genes or for OG-induced resistance to B. cinerea, whereas callose accumulation requires a functional AtrbohD. OG-induced resistance to B. cinerea is also unaffected in powdery mildew resistant4, despite the fact that callose accumulation was almost abolished in this mutant. These results indicate that the OG-induced oxidative burst is not required for the activation of defense responses effective against B. cinerea, leaving open the question of the role of reactive oxygen species in elicitor-mediated defense.     

Analysis of the Secretomes of Paracoccidioides Mycelia and Yeast Cells

Paracoccidioides, a complex of several phylogenetic species, is the causative agent of paracoccidioidomycosis. The ability of pathogenic fungi to develop a multifaceted response to the wide variety of stressors found in the host environment is important for virulence and pathogenesis. Extracellular proteins represent key mediators of the host-parasite interaction. To analyze the expression profile of the proteins secreted by Paracoccidioides, Pb01 mycelia and yeast cells, we used a proteomics approach combining two-dimensional electrophoresis with matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS/MS). From three biological replicates, 356 and 388 spots were detected, in mycelium and yeast cell secretomes, respectively. In this study, 160 non-redundant proteins/isoforms were indentified, including 30 and 24 proteins preferentially secreted in mycelia and yeast cells, respectively. In silico analyses revealed that 65% of the identified proteins/isoforms were secreted primarily via non-conventional pathways. We also investigated the influence of protein export inhibition in the phagocytosis of Paracoccidioides by macrophages. The addition of Brefeldin A to the culture medium significantly decreased the production of secreted proteins by both Paracoccidioides and internalized yeast cells by macrophages. In contrast, the addition of concentrated culture supernatant to the co-cultivation significantly increased the number of internalized yeast cells by macrophages. Importantly, the proteins detected in the fungal secretome were also identified within macrophages. These results indicate that Paracoccidioides extracellular proteins are important for the fungal interaction with the host.