Pseudocercospora fijiensis Conidial Germination Is Dominated by Pathogenicity Factors and Effectors.

Conidia play a vital role in the survival and rapid spread of fungi. Many biological processes of conidia, such as adhesion, signal transduction, the regulation of oxidative stress, and autophagy, have been well studied. In contrast, the contribution of pathogenicity factors during the development of conidia in fungal phytopathogens has been poorly investigated. To date, few reports have centered on the pathogenicity functions of fungal phytopathogen conidia. Pseudocercospora fijiensis is a hemibiotrophic fungus and the causal agent of the black Sigatoka disease in bananas and plantains. Here, a conidial transcriptome of P. fijiensis was characterized computationally. Carbohydrates, amino acids, and lipid metabolisms presented the highest number of annotations in Gene Ontology. Common conidial functions were found, but interestingly, pathogenicity factors and effectors were also identified. Upon analysis of the resulting proteins against the Pathogen-Host Interaction (PHI) database, 754 hits were identified. WideEffHunter and EffHunter effector predictors identified 618 effectors, 265 of them were shared with the PHI database. A total of 1107 conidial functions devoted to pathogenesis were found after our analysis. Regarding the conidial effectorome, it was found to comprise 40 canonical and 578 non-canonical effectors. Effectorome characterization revealed that RXLR, LysM, and Y/F/WxC are the largest effector families in the P. fijiensis conidial effectorome. Gene Ontology classification suggests that they are involved in many biological processes and metabolisms, expanding our current knowledge of fungal effectors.

Moonlight-like proteins are actually cell wall components in Pseudocercospora fijiensis.

The fungal cell wall protects fungi against threats, both biotic and abiotic, and plays a role in pathogenicity by facilitating host adhesion, among other functions. Although carbohydrates (e.g. glucans, chitin) are the most abundant components, the fungal cell wall also harbors ionic proteins, proteins bound by disulfide bridges, alkali-extractable, SDS-extractable, and GPI-anchored proteins, among others; the latter consisting of suitable targets which can be used for fungal pathogen control. Pseudocercospora fijiensis is the causal agent of black Sigatoka disease, the principal threat to banana and plantain worldwide. Here, we report the isolation of the cell wall of this pathogen, followed by extensive washing to eliminate all loosely associated proteins and conserve those integrated to its cell wall. In the HF-pyridine protein fraction, one of the most abundant protein bands was recovered from SDS-PAGE gels, electro-eluted and sequenced. Seven proteins were identified from this band, none of which were GPI-anchored proteins. Instead, atypical (moonlight-like) cell wall proteins were identified, suggesting a new class of atypical proteins, bound to the cell wall by unknown linkages. Western blot and histological analyses of the cell wall fractions support that these proteins are true cell wall proteins, most likely involved in fungal pathogenesis/virulence, since they were found conserved in many fungal pathogens.

A computationally simplistic poly-phasic approach to explore microbial communities from the Yucatan aquifer as a potential sources of novel natural products.

The need for new antibiotics has sparked a search for the microbes that might potentially produce them. Current sequencing technologies allow us to explore the biotechnological potential of microbial communities in diverse environments without the need for cultivation, benefitting natural product discovery in diverse ways. A relatively recent method to search for the possible production of novel compounds includes studying the diverse genes belonging to polyketide synthase pathways (PKS), as these complex enzymes are an important source of novel therapeutics. In order to explore the biotechnological potential of the microbial community from the largest underground aquifer in the world located in the Yucatan, we used a polyphasic approach in which a simple, non-computationally intensive method was coupled with direct amplification of environmental DNA to assess the diversity and novelty of PKS type I ketosynthase (KS) domains. Our results suggest that the bioinformatic method proposed can indeed be used to assess the novelty of KS enzymes; nevertheless, this in silico study did not identify some of the KS diversity due to primer bias and stringency criteria outlined by the metagenomics pipeline. Therefore, additionally implementing a method involving the direct cloning of KS domains enhanced our results. Compared to other freshwater environments, the aquifer was characterized by considerably less diversity in relation to known ketosynthase domains; however, the metagenome included a family of KS type I domains phylogenetically related, but not identical, to those found in the curamycin pathway, as well as an outstanding number of thiolases. Over all, this first look into the microbial community found in this large Yucatan aquifer and other fresh water free living microbial communities highlights the potential of these previously overlooked environments as a source of novel natural products.

Identification and in silico characterization of two novel genes encoding peptidases S8 found by functional screening in a metagenomic library of Yucatán underground water.

Metagenomics is a culture-independent technology that allows access to novel and potentially useful genetic resources from a wide range of unknown microorganisms. In this study, a fosmid metagenomic library of tropical underground water was constructed, and clones were functionally screened for extracellular proteolytic activity. One of the positive clones, containing a 41,614-bp insert, had two genes with 60% and 68% identity respectively with a peptidase S8 of Chitinimonas koreensis. When these genes were individually sub-cloned, in both cases their sub-clones showed proteolytic phenotype, confirming that they both encode functional proteases. These genes -named PrAY5 and PrAY6- are next to each other. They are similar in size (1845bp and 1824bp respectively) and share 66.5% identity. An extensive in silico characterization showed that their ORFs encode complex zymogens having a signal peptide at their 5' end, followed by a pro-peptide, a catalytic region, and a PPC domain at their 3' end. Their translated sequences were classified as peptidases S8A by sequence comparisons against the non-redundant database and corroborated by Pfam and MEROPS. Phylogenetic analysis of the catalytic region showed that they encode novel proteases that clustered with the sub-family S8_13, which according to the CDD database at NCBI, is an uncharacterized subfamily. They clustered in a clade different from the other three proteases S8 found so far by functional metagenomics, and also different from proteases S8 found in sequenced environmental samples, thereby expanding the range of potentially useful proteases that have been identified by metagenomics. I-TASSER modeling corroborated that they may be subtilases, thus possibly they participate in the hydrolysis of proteins with broad specificity for peptide bonds, and have a preference for a large uncharged residue in P1.

Yucatán in black and red: Linking edaphic analysis and pyrosequencing-based assessment of bacterial and fungal community structures in the two main kinds of soil of Yucatán State.

Yucatán State is dominated by two kinds of soil, named "Black Leptosol" and "Red Leptosol", which are interwoven across the State. In this work, we analyzed the relation between the edaphic characteristics and the bacterial and fungal community structures in these two kinds of Leptosol. The results revealed that Black Leptosol (BlaS) had a higher content of calcium carbonates, organic matter, nitrogen, and phosphorus than Red Leptosol (RedS). The most outstanding difference in the bacterial community structure between BlaS and RedS was that while in BlaS Actinobacteria was the most abundant phylum (43.7%), followed by Acidobacteria (26.9%) and Proteobacteria (23.6%), in RedS the bacterial community was strongly dominated by Acidobacteria (83%). Two fungal phyla were identified in both kinds of soil; Ascomycota, with 77% in BlaS and 56% in RedS, and Basidiomycota, with 22% in RedS and only 0.67% in BlaS. The most relevant difference between the two fungal communities was that excepting for Fusarium sp., all the species they had were different. Thus, in contrast with bacterial communities, where most of the major OTUs were present in both kinds of soil, fungal communities appeared to be unique to each kind of Leptosol.

Differential effects of aluminum on in vitro primary root growth, nutrient content and phospholipase C activity in coffee seedlings (Coffea arabica).

Coffea arabica is a woody species that grows in acid soils, where aluminum is available and may affect growth and productivity. To determine the effect of aluminum on primary root growth of C. arabica cv. Typica, seedlings were exposed over 30 days to different concentrations of AlCl3 (0, 100, 300 and 500 µM) in vitro. The aluminum effect on primary root growth was dose-dependent: low aluminum concentrations (100 and 300 µM) stimulated primary root growth (6.98 ± 0.15 and 6.45 ± 0.17 cm, respectively) compared to the control (0 µM; 5.24 ± 0.17 cm), while high concentrations (500 µM) induced damage to the root tips and inhibition of primary root growth (2.96 ± 0.28 cm). Aluminum (100 µM) also increased the K and Ca contents around 33% and 35% in the coffee roots. It is possible that aluminum toxicity resides in its association with cell nuclei in the meristematic region of the root. Additionally, after 30 days of treatment with aluminum, two different effects could be observed on phospholipase C (PLC) activity. In shoots, aluminum concentrations ≥ 300 µM inhibited more than 50% of PLC activity. In contrast, in roots a contrasting behavior was determined: low (100 µM) and toxic concentrations (500 µM) increased the activity of PLC (100%). These results suggest the possible involvement of the phosphoinositide signal transduction pathway, with the phospholipase C enzyme participating in the beneficial and toxic effects of aluminum in plants.

Acidobacteria appear to dominate the microbiome of two sympatric Caribbean Sponges and one Zoanthid

BACKGROUND: Marine invertebrate-associated microbial communities are interesting examples of complex symbiotic systems and are a potential source of biotechnological products. RESULTS: In this work, pyrosequencing-based assessment from bacterial community structures of sediments, two sponges, and one zoanthid collected in the Mexican Caribbean was performed. The results suggest that the bacterial diversity at the species level is higher in the sediments than in the animal samples. Analysis of bacterial communities' structure showed that about two thirds of the bacterial diversity in all the samples belongs to the phyla Acidobacteria and Proteobacteria. The genus Acidobacteriumappears to dominate the bacterial community in all the samples, reaching almost 80% in the sponge Hyrtios. CONCLUSIONS: Our evidence suggests that the sympatric location of these benthonic species may lead to common bacterial structure features among their bacterial communities. The results may serve as a first insight to formulate hypotheses that lead to more extensive studies of sessile marine organisms' microbiomes from the Mexican Caribbean.

GTPgammaS antagonizes the mastoparan-induced in vitro activity of PIP-phospholipase C from symbiotic root nodules of Phaseolus vulgaris.

Phospholipase C (PLC) has been suggested to have a role in signal perception by Nod factors (NFs) in legume root hair cells. For instance, mastoparan, a well-described agonist of heterotrimeric G protein, induces nodulin expression after NFs treatment or Rhizobium inoculation. Furthermore, it has been recently demonstrated that mastoparan also mimics calcium oscillations induced by NFs, suggesting that PLC could play a key role during the nodulation process. In this study, we elucidate a biochemical relationship between PLC and heterotrimeric G proteins during NFs signaling in legumes. In particular, the effect of NFs on in vitro PLC activity from nodule membrane fractions in the presence of guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) and mastoparan was assayed. Our results indicate that for phosphatidylinositol 4,5 bisphosphate (PIP(2))-PLC, there is a specific activity of 20-27 nmol mg(-1) min(-1) in membrane fractions of nodules 18-20 days after inoculation with Rhizobium tropici. Interestingly, in the presence of 5 microM mastoparan, PIP(2)-PLC activity was almost double the basal level. In contrast, PIP(2)-PLC activity was downregulated by 1-10 microM GTPgammaS. Also, PLC activity was decreased by up to 64% in the presence of increasing concentrations of NFs (10(-8) to 10(-5) M). NFs are critical signaling molecules in rhizobia/legume symbiosis that can activate many of the plant's early responses during nodule development. Calcium spiking, kinases, PLC activity and possibly G proteins appear to be components downstream of the NFs perception pathway. Our results suggest the occurrence of a dual signaling pathway that could involve both G proteins and PLC in Phaseolus vulgaris during the development of root nodules.

Isolation of cDNA encoding the catalytic site of phosphatidylinositol-specific phospholipase C from Coffea arabica L.: Recombinant expression and peptide purification.

A cDNA encoding the catalytic site of a phosphatidylinositol-specific phospholipase C (PI-PLC) was isolated from Coffea arabica suspension cells. The cDNA (designated CaPLC) encodes a polypeptide of 308 amino acids, containing the catalytic X and Y domains, and has 99% identity to the soybean gene. Recombinant CaPLC protein was expressed in Escherichia coli, purified, and used to produce a polyclonal antibody. The peptide has a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses. Immunoblots revealed the presence of PLC-like proteins in the tissues of different plant species.

Membrane-associated phosphoinositides-specific phospholipase C forms from Catharanthus roseus transformed roots.

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membraneassociated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.

The biochemical basis for the resistance to aluminum and their potential as selection markers.

Aluminum (Al) toxicity is one of the most widespread agronomic problems in world agriculture. The cellular mechanisms that some plant species use to tolerate Al are still not understood today. This knowledge is essential in order to develop crop species that can be cultivated on acid soils. Plant cell culture has been used for the investigation of both: Al-toxicity and -tolerance. The method used to obtain Al-tolerant cell line will be shown; as well as a protocol to measure Al-free concentration in the culture medium by using Morin (a fluorescent histochemical indicator for Al). Because Al affects different processes involved in the signal transduction pathway such as phospholipase C (PLC) activity, which could serve as selection marker, the PLC activity determination will be introduced.