Secretome analysis of the phytopathogen Macrophomina phaseolina cultivated in liquid medium supplemented with and without soybean leaf infusion.

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen that causes root and stem rot in several economically important crops. However, most of disease control strategies have shown limited effectiveness. Despite its impact on agriculture, molecular mechanisms involved in the interaction with host plant remains poorly understood. Nevertheless, it has been proven that fungal pathogens secrete a variety of proteins and metabolites to successfully infect their host plants. In this study, a proteomic analysis of proteins secreted by M. phaseolina in culture media supplemented with soybean leaf infusion was performed. A total of 250 proteins were identified with a predominance of hydrolytic enzymes. Plant cell wall degrading enzymes together peptidases were found, probably involved in the infection process. Predicted effector proteins were also found that could induce plant cell death or suppress plant immune response. Some of the putative effectors presented similarities to known fungal virulence factors. Expression analysis of ten selected protein-coding genes showed that these genes are induced during host tissue infection and suggested their participation in the infection process. The identification of secreted proteins of M. phaseolina could be used to improve the understanding of the biology and pathogenesis of this fungus. Although leaf infusion was able to induce changes at the proteome level, it is necessary to study the changes induced under conditions that mimic the natural infection process of the soil-borne pathogen M. phaseolina to identify virulence factors.

Identification of reference genes and their validation for gene expression analysis in phytopathogenic fungus Macrophomina phaseolina.

Macrophomina phaseolina is a soil-borne pathogenic fungus that infects a wide range of crop species and causes severe yield losses. Although the genome of the fungus has been sequenced, the molecular basis of its virulence has not been determined. Identification of up-regulated genes during fungal infection is important to understand the mechanism involved in its virulence. To ensure reliable quantification, expression of target genes needs to be normalized on the basis of certain reference genes. However, in the case of M. phaseolina, reference genes or their expression analysis have not been reported in the literature. Therefore, the objective of this study was to evaluate 12 candidate reference genes for the expression analysis of M. phaseolina genes by applying three different fungal growth conditions: a) during root and stem infection of soybean, b) in culture media with and without soybean leaf infusion and c) by inoculating a cut-stem. Based on BestKeeper, geNorm and NormFinder algorithms, CYP1 was identified as the best recommended reference gene followed by EF1ß for expression analysis of fungal gene during soybean root infection. Besides Mp08158, CYP1 gene was found suitable when M. phaseolina was grown in potato-dextrose broth with leaf infusion. In the case of cut-stem inoculation, Mp08158 and Mp11185 genes were found to be most stable. To validate the selected reference genes, expression analysis of two cutinase genes was performed. In general, the expression patterns were similar when the target genes were normalized against most or least stable gene. However, in some cases different expression pattern can be obtained when least stable gene is used for normalization. We believe that the reference genes identified and validated in this study will be useful for gene expression analysis during host infection with M. phaseolina.

Lathyrane Diterpenes from the Latex of Euphorbia laurifolia.

Two new macrocyclic diterpenes, 2-epi-latazienone (4) and 15ß-acetoxy-7ß-nicotinoyloxy-3ß,8α-di-(2-methylpropanoyloxy)-4αH,9αH,l1αH-lathyra- 5E,12E-dien-14-one (5), and three known lathyrane-type diterpenes (1-3) were isolated from Euphorbia laurifolia latex. Their structures were determined on the basis of a detailed analysis of their 1D and 2D NMR spectroscopic and mass spectral data.