Selection of optimized candidate reference genes for qRT-PCR normalization in rice (Oryza sativa L.) during Magnaporthe oryzae infection and drought.

Drought and rice blast disease caused by Magnaporthe oryzae are two of the most serious threats to global rice production. To explore the mechanisms underlying gene expression induced in rice by stresses, studies involving transcriptome analyses have been conducted over the past few years. Thus, it is crucial to have a reliable set of reference genes to normalize the expression levels of rice genes affected by different stresses. To identify potential reference genes for studies of the differential expression of target genes in rice under M. oryzae infection and drought conditions, the present study evaluated five housekeeping genes for the normalization of gene expression. The stability of the expression of these genes was assessed using the analytical software packages geNorm and NormFinder. For all samples analyzed, the stability rank was UBQ5 > GAPDH > eIF-4α> ß-TUB > 18S rRNA. The data showed that the UBQ5, GAPDH, and eIF-4αgenes are appropriate, high-performing reference genes and will be highly useful in future expression studies of fungal infections and drought in rice.

Morpho-anatomical characterization of mature embryo-derived callus of rice (Oryza sativa L.) suitable for transformation.

The objective of this study was to morpho-anatomically characterize embryogenic rice calli during early induction of somatic embryogenesis of three Brazilian rice cultivars. Herein, we explored embryogenic units (EUs) from 2-week-old cut proliferated calli to verify whether they were suitable for Agrobacterium tumefasciens-mediated transformation. Histological analysis and scanning electron microscopy (SEM) were used to analyze these types of calli during early rice callogenesis in the cultivars BRS Primavera, BRS Bonança, and BRS Caiapó. The characteristics of the embryogenic cells were preserved in the EUs, which showed a globular, compact structure that contained tightly packed cells and thus rendered the cells suitable for transformation. The EUs of BRS Caiapó also maintained the characteristics of the non-embryogenic callus, such as an elongated morphology and a lack of cellular organization. In general, the observations of the histological sections corresponded with those of the SEM images. The histological analysis suggested that all cultivars used in these experiments have morphogenic potential. The EUs from proliferated 2-week-old cut calli maintained their embryogenic features. The EUs were subjected to Agrobacterium-mediated transformation, which exhibited a regeneration frequency of 58 % for transformed hygromycin-resistant cell lines. These results show that EUs from proliferated 2-week-old cut calli are suitable for plant transformation.

Comparative in vitro and in planta analyses of extracellular enzymes secreted by the pathogenic fungus Sclerotinia sclerotiorum.

Dry bean (Phaseolus vulgaris L.) is an important economic crop in Brazil but its yield can be significantly reduced by white mold, a disease caused by Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic, highly destructive, and non-host-specific fungus. This fungus secretes numerous cell wall-degrading enzymes such as polygalacturonases, exo-ß-1,3-glucanases, xylanases, and cellulases that have been detected during the early stages of infection. In this study, the activities of these enzymes were detected in all carbon sources tested (citrus pectin, cell wall extract from P. vulgaris, carboxymethyl cellulose, and glucose), but the highest levels were found when using citrus pectin and cell wall extract from P. vulgaris. Regardless of the carbon source, pH decreased throughout the culture time. During pathogenesis in dry bean stems, increased enzyme activities were also observed. Reverse transcriptase-polymerase chain reaction experiments showed that the induction of polygalacturonases (sspg1, sspg3, sspg5, sspg6, and sspg7), exo-ß-1,3-glucanases, and endo-ß-1-4-glucanase in S. sclerotiorum occurred during the early stages of colonization.

Effect of different carbon sources on proteases secreted by the fungal pathogen Sclerotinia sclerotiorum during Phaseolus vulgaris infection.

Sclerotinia sclerotiorum (Sclerotiniaceae) is a plant pathogenic fungus that causes white mold disease in vegetable crops, including the common bean (Phaseolus vulgaris). Proteases produced by fungi are normally an important part of the pathogenic process in the host. We examined the effect of different carbon sources--pectin, glucose, and cell wall of P. vulgaris on the production of proteases in cultures of S. sclerotiorum. These proteases were also assayed in infected P. vulgaris plants. Enzyme activity was increased with all carbon sources, but the highest levels were found when pectin was added. Based on real-time quantitative reverse transcription-PCR analyses, protease induction in S. sclerotiorum was found to occur at the level of gene transcription. The finding of increased expression of acid phosphatase 1 and aspartyl protease in vivo in infected P. vulgaris plants supports the role of these enzymes in the invasion process of S. sclerotiorum.

Genetic diversity and mycelial compatibility groups of the plant-pathogenic fungus Sclerotinia sclerotiorum in Brazil.

The genetic variability of 40 Sclerotinia sclerotiorum isolates from various fields widely distributed throughout Brazil and different host crops was analyzed using RAPD markers and mycelial compatibility groupings (MCGs). The isolates were characterized using 16 random primers of the OPERON series, which produced 121 DNA fragments. UPGMA cluster analysis using Jaccard's genetic distance and MCGs allowed separation of the isolates into three clusters, with similarity indices of 68.2, 61.8, and 61.8%, and five MCGs. The haplotypes obtained with RAPD markers provided very characteristic groupings of S. sclerotiorum isolates according to MCG, but did not show any relationship with geographic origin or host type. Furthermore, analysis of molecular variance demonstrated that 99.1% of the observed variation was a result of genetic differences between individuals; the host culture did not have a significant effect. This is the first report of high level variability of S. sclerotiorum in Brazil based on the study of isolates of wide geographical origin, supported by RAPD markers and MCGs. These results endorse the prevalence of sexual reproduction in tropical and subtropical regions in contrast to clonal reproduction in temperate regions.

Characterization of the dry bean polygalacturonase-inhibiting protein (PGIP) gene family during Sclerotinia sclerotiorum (Sclerotiniaceae) infection.

Polygalacturonase-inhibiting proteins are leucine-rich repeat proteins that inhibit fungal endopolygalacturonases. The interaction of polygalacturonase-inhibiting protein with endopolygalacturonases limits the destructive potential of endopolygalacturonases and may trigger plant defense responses induced by oligogalacturonides. We examined the expression of fungal pg and plant Pvpgip genes in bean (Phaseolus vulgaris) stems infected with Sclerotinia sclerotiorum to determine whether any of them are associated with the infection process. Transcriptional analysis was carried out by means of semi-quantitative reverse transcription PCR or real-time PCR. The sspg1 gene was highly expressed during infection; sspg3 was regulated during the later phases of infection; sspg5 was more uniformly expressed during infection, whereas sspg6 was only weakly expressed. During the course of infection, Pvpgip1 transcripts were not detected at early stages, but they appeared 72 h post-inoculation. High levels of Pvpgip2 expression were observed during the initial phase of infection; the transcript peaked by 48 h post-inoculation and declined by 72 h post-inoculation. Pvpgip3 expression increased strongly at 96 h post-inoculation. Pvpgip4 was constantly present from 24 h post-inoculation until the end of the experiment. However, we detected higher levels of the Pvpgip4 transcript in the necrotic lesion area than in plants that had been mechanically wounded. Remarkably, only Pvpgip4 appeared to be moderately induced by mechanical wounding. These results provide evidence that endopolygalacturonases contribute to the infection process during host colonization by promoting the release of plant cell oligogalacturonides, which are powerful signaling molecules and may also activate plant defenses, such as polygalacturonase-inhibiting proteins.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis.

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.