BackgroundThe Hsp20 genes are associated with stress caused by HS and other abiotic factors, but have recently been found to be associated with the response to biotic stresses. These genes represent the most abundant class among the HSPs in plants, but little is known about this gene family in soybean. Because of their apparent multifunctionality, these proteins are promising targets for developing crop varieties that are better adapted to biotic and abiotic stresses. Thus, in the present study an in silico identification of GmHsp20 gene family members was performed, and the genes were characterized and subjected to in vivo expression analysis under biotic and abiotic stresses.ResultsA search of the available soybean genome databases revealed 51 gene models as potential GmHsp20 candidates. The 51 GmHsp20 genes were distributed across a total of 15 subfamilies where a specific predicted secondary structure was identified. Based on in vivo analysis, only 47 soybean Hsp20 genes were responsive to heat shock stress. Among the GmHsp20 genes that were potentials HSR, five were also cold-induced, and another five, in addition to one GmAcd gene, were responsive to Meloidogyne javanica infection. Furthermore, one predicted GmHsp20 was shown to be responsive only to nematode infection; no expression change was detected under other stress conditions. Some of the biotic stress-responsive GmHsp20 genes exhibited a divergent expression pattern between resistant and susceptible soybean genotypes under M. javanica infection. The putative regulatory elements presenting some conservation level in the GmHsp20 promoters included HSE, W-box, CAAT box, and TA-rich elements. Some of these putative elements showed a unique occurrence pattern among genes responsive to nematode infection.ConclusionsThe evolution of Hsp20 family in soybean genome has most likely involved a total of 23 gene duplications. The obtained expression profiles revealed that the majority of the 51 GmHsp20 candidates are induced under HT, but other members of this family could also be involved in normal cellular functions, unrelated to HT. Some of the GmHsp20 genes might be specialized to respond to nematode stress, and the predicted promoter structure of these genes seems to have a particular conserved pattern related to their biological function.
Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales- or P. pachyrhizi-specific families (tribes). Members of some families were strongly up-regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors.
Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) causes bacterial wilt on beans (Phaseolus vulgaris) and bacterial tan spot on soybeans (Glycine max). Cff was detected on beans in Brazil in 1995. Plants of commercial and experimental fields of soybean with typical symptoms of the disease were collected in the State of Paraná, Brazil, during the 2011/2012 growing season. The causal agent was identified as Cff by isolation from symptomatic leaves on CNS semi-selective medium, artificial inoculation test and re-isolation in soybean and bean, Gram staining test, solubility in KOH, and by PCR. This is the first report of Cff on soybean in Brazil.
Rust fungi are devastating pathogens for several important crop plants. The biotrophic lifestyle of rust fungi requires that they influence their host plants to create a favorable environment for growth and reproduction. Rust fungi secrete a variety of effector proteins that manipulate host target proteins to alter plant metabolism and suppress defense responses. Because of the obligate biotrophic lifestyle of rust fungi, direct evidence for effector function is difficult to obtain, and so suites of experiments utilizing expression in heterologous systems are necessary. Here, we present results from a yeast cell death suppression assay and assays for suppression of PAMP-triggered immunity (PTI) and effector triggered immunity (ETI) based on delivery of effectors through the bacterial type III secretion system. In addition, subcellular localization was tested using transient expression of GFP fusion proteins in Nicotiana benthamiana through Agrobacterium infiltration. We tested 31 representative effector candidates from the devastating common bean rust pathogen Uromyces appendiculatus. These effector candidates were selected based on features of their gene families, most important lineage specificity. We show that several of our effector candidates suppress plant defense. Some of them also belong to families of effector candidates that are present in multiple rust species where their homologs probably also have effector functions. In our analysis of candidate effector mRNA expression, some of those effector candidates that gave positive results in the other assays were not up-regulated during plant infection, indicating that either these proteins have functions at multiple life stages or that strong up-regulation of RNA level in planta may not be as important a criterion for identifying effectors as previously thought. Overall, our pipeline for selecting effector candidates based on sequence features followed by screening assays using heterologous expression systems was successful in discriminating effector candidates. This work lays the foundation for functional characterization of U. appendiculatus effectors, the identification of effector targets, and identification of novel sources for resistance in common bean.
Asian soybean rust, caused by the fungus Phakopsora pachyrhizi, is one of the most important diseases affecting soybean production in tropical areas. During infection, P. pachyrhizi secretes proteins from haustoria that are transferred into plant cells to promote virulence. To date, only one candidate P. pachyrhizi effector protein has been characterized in detail to understand the mechanism by which it suppresses plant defenses to enhance infection. Here, we aimed to extend understanding of the pathogenic mechanisms of P. pachyrhizi based on the discovery of host proteins that interact with the effector candidate Phapa-7431740. We demonstrated that Phapa-7431740 suppresses pathogen-associated molecular pattern triggered immunity (PTI), and it interacts with a soybean glucan endo-1,3- β-glucosidase (GmβGLU), a pathogenesis-related (PR) protein belonging to the PR-2 family. Structural and phylogenetic characterization of PR-2 protein family predicted in the soybean genome and comparison to PR-2 family members in Arabidopsis thaliana and cotton, demonstrated that GmβGLU is a type IV β-1,3-glucanase. Transcriptional profiling during an infection time course showed that the GmβGLU mRNA is highly induced during the initial hours after infection, coinciding with peak of expression of Phapa-7431740. The effector was able to interfere with the activity of GmβGLU in vitro, with a dose-dependent inhibition. Our results suggest that Phapa-7431740 may suppress PTI by interfering with glucan endo-1,3-β-glucosidase activity.
Asian soybean rust (ASR) is one of the most destructive diseases affecting soybeans. The causative agent of ASR, the fungus Phakopsora pachyrhizi, presents characteristics that make it difficult to study in vitro, limiting our knowledge of plant-pathogen dynamics. Therefore, this work used leaf lesion laser microdissection associated with deep sequencing to determine the pathogen transcriptome during compatible and incompatible interactions with soybean. The 36,350 generated unisequences provided an overview of the main genes and biological pathways that were active in the fungus during the infection cycle. We also identified the most expressed transcripts, including sequences similar to other fungal virulence and signaling proteins. Enriched P. pachyrhizi transcripts in the resistant (PI561356) soybean genotype were related to extracellular matrix organization and metabolic signaling pathways and, among infection structures, in amino acid metabolism and intracellular transport. Unisequences were further grouped into gene families along predicted sequences from 15 other fungi and oomycetes, including rust fungi, allowing the identification of conserved multigenic families, as well as being specific to P. pachyrhizi. The results revealed important biological processes observed in P. pachyrhizi, contributing with information related to fungal biology and, consequently, a better understanding of ASR.
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