Use of AFLP markers to estimate molecular diversity of Phakopsora pachyrhizi

Background Asian soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. & Syd., is one of the main diseases affecting soybean and has been reported as one of the most economically important fungal pathogens worldwide. Knowledge of the genetic diversity of this fungus should be considered when developing resistance breeding strategies. We aimed to analyze the genetic diversity of P. pachyrhizi combining simple sampling with a powerful and reproducible molecular technique. Results We employed Amplified Fragment Length Polymorphism (AFLP) technique for the amplification of P. pachyrhizi DNA extracted from naturally SBR-infected plants from 23 production fields. From a total of 1919 markers obtained, 77% were polymorphic. The high percentage of polymorphism and the Nei's genetic diversity coefficient (0.22) indicated high pathogen diversity. Analysis of molecular variance showed higher genetic variation within countries than among them. Temporal analysis showed a higher genetic variation within a year than between years. Cluster, phylogenetic and principal co-ordinate analysis showed that samples group by year of collection and then by country sampled. Conclusions The study proposed combining a simple collection of urediniospore with a subsequent analysis by AFLP was useful to examine the molecular polymorphism of samples of P. pachyrhizi collected and might have a significant contribution to the knowledge of its genetic diversity. Also, AFLP analysis is an important and potent molecular tool for the study of genetic diversity and could be useful to carry out wider genetic diversity studies.

Identification of the soybean HyPRP family and specific gene response to Asian soybean rust disease.

Soybean [Glycine max (L.) Merril], one of the most important crop species in the world, is very susceptible to abiotic and biotic stress. Soybean plants have developed a variety of molecular mechanisms that help them survive stressful conditions. Hybrid proline-rich proteins (HyPRPs) constitute a family of cell-wall proteins with a variable N-terminal domain and conserved C-terminal domain that is phylogenetically related to non-specific lipid transfer proteins. Members of the HyPRP family are involved in basic cellular processes and their expression and activity are modulated by environmental factors. In this study, microarray analysis and real time RT-qPCR were used to identify putative HyPRP genes in the soybean genome and to assess their expression in different plant tissues. Some of the genes were also analyzed by time-course real time RT-qPCR in response to infection by Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease. Our findings indicate that the time of induction of a defense pathway is crucial in triggering the soybean resistance response to P. pachyrhizi. This is the first study to identify the soybean HyPRP group B family and to analyze disease-responsive GmHyPRP during infection by P. pachyrhizi.

Subtractive libraries for prospecting differentially expressed genes in the soybean under water deficit.

Soybean has a wide range of applications in the industry and, due to its crop potential, its improvement is widely desirable. During drought conditions, soybean crops suffer significant losses in productivity. Therefore, understanding the responses of the soybean under this stress is an effective way of targeting crop improvement techniques. In this study, we employed the Suppressive Subtractive Hybridization (SSH) technique to investigate differentially expressed genes under water deficit conditions. Embrapa 48 and BR 16 soybean lines, known as drought-tolerant and -sensitive, respectively, were grown hydroponically and subjected to different short-term periods of stress by withholding the nutrient solution. Using this approach, we have identified genes expressed during the early response to water deficit in roots and leaves. These genes were compared among the lines to assess probable differences in the plant transcriptomes. In general, similar biochemical processes were predominant in both cultivars; however, there were more considerable differences between roots and leaves of Embrapa 48. Moreover, we present here a fast, clean and straightforward method to obtain drought-stressed root tissues and a large enriched collection of transcripts expressed by soybean plants under water deficit that can be useful for further studies towards the understanding of plant responses to stress.

Ubiquitous urease affects soybean susceptibility to fungi.

The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach.

Size of AT(n) insertions in promoter region modulates Gmhsp17.6-L mRNA transcript levels.

During earlier experiments, an SSR molecular marker (176 Soy HSP) showing high correlation (70%) with resistance/susceptibility to javanese root-knot nematode Meloidogyne javanica was identified in soybean. After being sequenced, results indicated that the SSR 176 Soy HSP marker was inserted in the promoter region of Gmhsp17.6-L gene. It was also detected in this region that resistant genotypes presented insertions between AT(31) and AT(33) in size and susceptible genotypes, AT(9). Gmhsp17.6-L gene coding region presented a perfect match in amino acid sequence in all soybean genotypes. A ribonuclease protection assay showed that Gmhsp17.6-L gene mRNA transcripts were present in all genotypes. A real-time relative quantification (qPCR) indicated in the resistant individuals higher mRNA transcripts levels, which presented in the sequencing more AT(n) insertions. These results suggest that the number of AT(n) insertions inside this promoter region could modulate up or down gene levels. Those findings can lead to the possibility of manipulating, between some limits, the mRNA transcripts levels using different sizes of AT(n) insertions.

The nodC, nodG, and glgX genes of Rhizobium tropici strain PRF 81.

Rhizobium tropici is a diazotrophic microsymbiont of common bean (Phaseolus vulgaris L.) that encompasses important but still poorly studied tropical strains, and a recent significant contribution to the knowledge of the species was the publication of a genomic draft of strain PRF 81, which revealed several novel genes [Pinto et al. Funct Int Gen 9:263-270, 2009]. In this study, we investigated the transcription of nodC, nodG, and glgX genes, located in the nod operon of PRF 81 strain, by reverse-transcription quantitative PCR. All three genes showed low levels of transcription when the cells were grown until exponential growth phase in the presence of common-bean-seed exudates or of the root nod-gene inducer naringenin. However, when cells at the exponential phase of growth were incubated with seed exudates, transcription occurred after only 5 min, and nodC, nodG, and glgX were transcribed 121.97-, 14.86-, and 50.29-fold more than the control, respectively, followed by a rapid decrease in gene transcription. Much lower levels of transcription were observed in the presence of naringenin; furthermore, maximum transcription required 8 h of incubation for all three genes. In light of these results, the mechanisms of induction of the nodulation genes by flavonoids are discussed.

Polymorphism of the bm86 gene in South American strains of the cattle tick Boophilus microplus.

Thirty Boophilus microplus strains from various geographic regions of Brazil, Argentina, Uruguay, Venezuela and Colombia were analyzed for the bm86 and bm95 gene. A fragment of cDNA of 794 base pairs of the parasite larvae, included between nucleotides 278-1071s, was amplified and cloned on the pGEM-T vector. Two random clones were sequenced for each population and the nucleotides 278-1071 and predicted amino acid sequences compared with the bm86 and bm95 genes. Variations from 1.76 to 3.65% were detected in the nucleotides sequence when compared with the homologous sequence of the bm86 gene and a 3.4-6.08% in the homologous amino acid sequence of the Bm86 protein. When the sequences obtained were compared with the bm95 gene, variations from 0.50 to 3.15% were detected. Variations from 1.14 to 4.56% were detected for the Bm95 protein homologous sequences in the deduced amino acid sequence. Only five of the 30 strains analyzed presented two different types of alleles expressed and the two alleles of the Alegre population and allele 1 of the Betim population were the most divergent of all those analyzed.