José Antonio Cervantes-Chávez

Regulatory role of the PKA pathway in dimorphism and mating in Yarrowia lipolytica

Previous studies on the dimorphic transition of Yarrowia lipolytica suggested opposite roles for MAPK and PKA pathways in this phenomenon. To obtain conclusive evidences for these opposite roles we isolated and disrupted the unique gene encoding the Pka catalytic subunit (TPK1). TPK1 was regulated only at the post-transcriptional level, with Pka activity increasing during yeast-like growth. tpk1 null mutants were viable and without growth defects, but more sensitive to different stress conditions. Deltatpk1 mutants were mating-deficient, and grew constitutively in the mycelial form, whereas Deltaste11 (Mapkkk-less)/Deltatpk1 double mutants grew in the yeast form, indicating that this is the default growth pattern of the fungus. Our data confirm that MAPK and PKA pathways operate in opposition during the dimorphic behavior of Y. lipolytica, but synergic in mating. These data stress the idea that in different fungi both signal transduction systems may operate distinctly or even be antagonistic or synergic in the coordination of cell responses to different stimuli.

Polyamine Metabolism in Fungi with Emphasis on Phytopathogenic Species

Polyamines are essential metabolites present in all living organisms, and this subject has attracted the attention of researchers worldwide interested in defining their mode of action in the variable cell functions in which they are involved, from growth to development and differentiation. Although the mechanism of polyamine synthesis is almost universal, different biological groups show interesting differences in this aspect that require to be further analyzed. For these studies, fungi represent interesting models because of their characteristics and facility of analysis. During the last decades fungi have contributed to the understanding of polyamine metabolism. The use of specific inhibitors and the isolation of mutants have allowed the manipulation of the pathway providing information on its regulation. During host-fungus interaction polyamine metabolism suffers striking changes in response to infection, which requires examination. Additionally the role of polyamine transporter is getting importance because of its role in polyamine regulation. In this paper we analyze the metabolism of polyamines in fungi, and the difference of this process with other biological groups. Of particular importance is the difference of polyamine biosynthesis between fungi and plants, which makes this process an attractive target for the control of phytopathogenic fungi.

Response to Environmental Stresses, Cell-wall Integrity, and Virulence Are Orchestrated Through the Calcineurin Pathway in Ustilago hordei

In eukaryotes, several biological processes are regulated through calcium signaling. Calcineurin is a calcium-calmodulin-regulated serine/threonine phosphatase consisting of catalytic subunit A and regulatory subunit B. Phosphatase activity resides in the catalytic subunit, which activates by dephosphorylation downstream components such as transcription factor Crz1. The importance of this pathway to respond to environmental stress has been explored in several fungal pathogens. The basidiomycete Ustilago hordei causes covered smut of barley. We addressed the role of the Ca(2+)-calcineurin activated pathway by deleting UhCna1 and UhCnb1. These genes were not essential in U. hordei but the corresponding mutants displayed a variety of phenotypes when applying environmental stress such as sensitivity to pH, temperature, H₂O₂, mono- and divalent cations; and to genotoxic, acid, or oxidative stresses. Cell-wall integrity was compromised and mutants displayed altered cell morphologies. Mating was delayed but not abolished, and combined sensitivities likely explained a severely reduced virulence toward barley plants. Expression analyses revealed that response to salt stress involved the induction of membrane ATPase genes UhEna1 and UhEna2, which were regulated through the calcineurin pathway. Upregulation of UhFKS1, a 1,3-β-d-glucan synthase gene, correlated with the increased amount of 1,3-β-d-glucan in the calcineurin mutants grown under salt stress.

A Trichoderma atroviride stress-activated MAPK pathway integrates stress and light signals.

Cells possess stress‐activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.

Phenotypic comparison of samdc and spe mutants reveals complex relationships of polyamine metabolism in Ustilago maydis

Synthesis of spermidine involves the action of two enzymes, spermidine synthase (Spe) and S-adenosylmethionine decarboxylase (Samdc). Previously we cloned and disrupted the gene encoding Spe as a first approach to unravel the biological function of spermidine in Ustilago maydis. With this background, the present study was designed to provide a better understanding of the role played by Samdc in the regulation of the synthesis of this polyamine. With this aim we proceeded to isolate and delete the gene encoding Samdc from U. maydis, and made a comparative analysis of the phenotypes of samdc and spe mutants. Both spe and samdc mutants behaved as spermidine auxotrophs, and were more sensitive than the wild-type strain to different stress conditions. However, the two mutants displayed significant differences: in contrast to spe mutants, samdc mutants were more sensitive to LiCl stress, high spermidine concentrations counteracted their dimorphic deficiency, and they were completely avirulent. It is suggested that these differences are possibly related to differences in exogenous spermidine uptake or the differential location of the respective enzymes in the cell. Alternatively, since samdc mutants accumulate higher levels of S-adenosylmethionine (SAM), whereas spe mutants accumulate decarboxylated SAM, the known opposite roles of these metabolites in the processes of methylation and differentiation offer an additional attractive hypothesis to explain the phenotypic differences of the two mutants, and provide insights into the additional roles of polyamine metabolism in the physiology of the cell.

Trehalose is required for stress resistance and virulence of the Basidiomycota plant pathogen Ustilago maydis

Trehalose is an important disaccharide that can be found in bacteria, fungi, invertebrates and plants. In some Ascomycota fungal plant pathogens, the role of trehalose was recently studied and shown to be important for conferring protection against several environmental stresses and for virulence. In most of the fungi studied, two enzymes are involved in the synthesis of trehalose: trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2). To study the role of trehalose in virulence and stress response in the Basidiomycota maize pathogen Ustilago maydis, Δtps2 deletion mutants were constructed. These mutants did not produce trehalose as confirmed by HPLC analysis, showing that the single gene disruption impaired its biosynthesis. The mutants displayed increased sensitivity to oxidative, heat, acid, ionic and osmotic stresses as compared to the wild-type strains. Virulence of Δtps2 mutants to maize plants was extremely reduced compared to wild-type strains, possibly due to reduced capability to deal with the hostile host environment. The phenotypic traits displayed by Δtps2 strains were fully restored to wild-type levels when complemented with the endogenous UmTPS2 gene, or a chimeric construct having the Saccharomyces cerevisiae TPS2 ORF. This report demonstrates the presence of a single biosynthetic pathway for trehalose, and its importance for virulence in this model Basidiomycota plant pathogen.

Isolation and molecular analysis of Umhda2 a gene encoding a histone deacetylase from Ustilago maydis.

By use of the polymerase chain reaction and synthetic oligonucleotides designed from conserved regions, we amplified a fragment of a gene from Ustilago maydis encoding a putative histone deacetylase. With this probe we isolated the full gene from a minigenomic library. The gene (designated as Umhda2) contains an open reading frame (ORF) of 1701 bp encoding a protein of 566 amino acids. Multiple comparison analysis with other histone deacetylases suggests that the Umhda2 gene product belongs to the Rpd3-related family of proteins. The highest degree of homology with histone deacetylases from other organisms corresponded to Hda1p of Schizosaccharomyces pombe and Rpd3p of Saccharomyces cerevisiae with 64.2 and 62.2% of sequence similarity, respectively. It displayed a substantially lower similarity with another histone deacetylase from U. maydis (Hda1p, 52.4%). Semi-quantitative RTPCR results indicate that the gene is transcriptionally up-regulated during the in  vitro yeast-to-mycelium dimorphic transition.

Phylogenetic Relationships and Acaricidal Effects of Beauveria bassiana Obtained from Cattle Farm Soils Against Rhipicephalus microplus

Abstract The objectives of the present study were to isolate Beauveria bassiana strains from cattle farm soils, analyze the phylogenetic relationships among the fungal strains isolated from these soils, and determine the acaricidal effect of B. bassiana isolates on engorged Rhipicephalus microplus tick strains resistant or susceptible to chemical acaricides. Six strains of B. bassiana were obtained and isolated from cattle farm soils in the Mexican tropics using the Galleria bait method, and their acaricidal effect was assessed against 2 populations of R. microplus (“Media Joya” chemical acaricide-resistant strain or “CLAR” chemical acaricide-susceptible strain) using the adult immersion test. The BbV03 strain produced 86.7% and 60% mortality in resistant and susceptible ticks on day 20, respectively, whereas the BbV04 strain produced 66.7% and 53.5% mortality in resistant and susceptible ticks on day 20, respectively. The BbV03 and BbV04 strains reduced egg laying on both R. microplus populations. There was no statistical difference in the acaricidal effect of B. bassiana strains among chemical acaricide-susceptible or -resistant R. microplus populations (P > 0.05). The BbV03 strain was the most virulent against R. microplus with an LC50 of 2 × 107 and LC99 of 7 × 108 conidia/ml. We found that the 6 B. bassiana isolated clustered in the same clade with other previously reported B. bassiana strains (from GenBank) but were separated into 3 different sub-clades. This study shows that some B. bassiana strains are a promising coadjuvant alternative for biological tick control, including tick populations that are resistant to chemical acaricides. Beauveria bassiana is present in the pastures of tropic cattle farms, and there are genetic variations between B. bassiana strains living in this ecosystem that might play an important role in the natural control of R. microplus in cattle farm paddocks.