MicroRNAs (miRNAs) are small RNA molecules recognized as important regulators of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data. We are interested in the identification of miRNAs in Phaseolus vulgaris (common bean) to uncover different plant strategies to cope with adverse conditions and because of its relevance as a crop in developing countries. Here we present the identification of conserved and candidate novel miRNAs in P. vulgaris present in different organs and growth conditions, including drought, abscisic acid treatment, and Rhizobium infection. We also identified cDNA sequences in public databases that represent the corresponding miRNA precursors. In addition, we predicted and validated target mRNAs amongst reported EST and cDNAs for P. vulgaris. We propose that the novel miRNAs present in common bean and other legumes, are involved in regulation of legume-specific processes including adaptation to diverse external cues.

This transformation procedure generates, with high efficiency (70-90%), hairy roots in cultivars, landraces and accessions of Phaseolus vulgaris (common bean) and other Phaseolus spp. Hairy roots rapidly develop after wounding young plantlets with Agrobacterium rhizogenes, at the cotyledon node, and keeping the plants in high-humidity conditions. Callogenesis always precedes hairy-root formation, and after 15 days, when roots develop at wounded sites, the stem with the normal root is cleaved below the hairy root zone. Transgenic roots and nodules co-transformed with a binary vector can be easily identified using a reporter gene. This procedure, in addition to inducing robust transgenic hairy roots that are susceptible to being nodulated by rhizobia and to fixing nitrogen efficiently, sets the foundation for a high-throughput functional genomics approach on the study of root biology and root-microbe interactions. This protocol can be completed within 30 days.

BackgroundMicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume Phaseolus vulgaris (common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of Phaseolus vulgaris.ResultsSmall RNA libraries were generated from roots, flowers, leaves, and seedlings of P. vulgaris. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies.ConclusionsThis work represents the first massive-scale RNA sequencing study performed in Phaseolus vulgaris to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of P. vulgaris miRNAs in relation to those of other legumes.

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.

Eukaryotes contain three types of lipid kinases that belong to the phosphatidylinositol 3-kinase (PI3K) family. In plants and Saccharomyces cerevisiae, only PI3K class III family members have been identified. These enzymes regulate the innate immune response, intracellular trafficking, autophagy, and senescence. Here, we report that RNAi-mediated downregulation of common bean (Phaseolus vulgaris) PI3K severely impaired symbiosis in composite P. vulgaris plants with endosymbionts such as Rhizobium tropici and Rhizophagus irregularis. Downregulation of Pv-PI3K was associated with a marked decrease in root hair growth and curling. Additionally, infection thread growth, root-nodule number, and symbiosome formation in root nodule cells were severely affected. Interestingly, root colonization by AM fungi and the formation of arbuscules were also abolished in PI3K loss-of-function plants. Furthermore, the transcript accumulation of genes encoding proteins known to interact with PI3K to form protein complexes involved in autophagy was drastically reduced in these transgenic roots. RNAimediated downregulation of one of these genes, Beclin1/Atg6, resulted in a similar phenotype as observed for transgenic roots in which Pv-PI3K had been downregulated. Our findings show that an autophagy-related process is crucial for the mutualistic interactions of P. vulgaris with beneficial microorganisms.