Genome-Wide Identification of Key Components of RNA Silencing in Two Phaseolus vulgaris Genotypes of Contrasting Origin and Their Expression Analyses in Response to Fungal Infection

RNA silencing serves key roles in a multitude of cellular processes, including development, stress responses, metabolism, and maintenance of genome integrity. Dicer, Argonaute (AGO), double-stranded RNA binding (DRB) proteins, RNA-dependent RNA polymerase (RDR), and DNA-dependent RNA polymerases known as Pol IV and Pol V form core components to trigger RNA silencing. Common bean (Phaseolus vulgaris) is an important staple crop worldwide. In this study, we aimed to unravel the components of the RNA-guided silencing pathway in this non-model plant, taking advantage of the availability of two genome assemblies of Andean and Meso-American origin. We identified six PvDCLs, thirteen PvAGOs, 10 PvDRBs, 5 PvRDRs, in both genotypes, suggesting no recent gene amplification or deletion after the gene pool separation. In addition, we identified one PvNRPD1 and one PvNRPE1 encoding the largest subunits of Pol IV and Pol V, respectively. These genes were categorized into subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization, and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different organs using online data and quantitative RT-PCR after pathogen infection. Several of the candidate genes were up-regulated after infection with the fungus Colletotrichum lindemuthianum.

Genome Wide Identification of Key Components of RNA Silencing in Two Phaseolus vulgaris Genotypes of Contrasting Origin and Their Expression Analyses in Response to Fungal Infection

RNA silencing serves key roles in a multitude of cellular processes, including development, stress responses, metabolism, and maintenance of genome integrity. Dicer, Argonaute (AGO), double-stranded RNA binding (DRB), RNA-dependent RNA polymerase (RDR) and DNA-dependent RNA polymerases known as Pol IV and Pol V form core components to trigger RNA silencing. Common bean (Phaseolus vulgaris) is an important staple crop worldwide. In this study, we aimed to unravel the components of the RNA-guided silencing pathway in this non-model plant taking advantage of the availability of two genome assemblies of Andean and Meso-American origin. We identified six PvDCLs, thirteen PvAGOs, 10 PvDRB, 5 PvRDR, in both genotypes, suggesting no recent gene amplification or deletion after the gene pool separation. In addition, we identified one PvNRPD1 and one PvNRPE1 encoding the largest subunits of Pol IV and Pol V, respectively. These genes were categorized into subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different organs using online data and quantitative RT-PCR after pathogen infection. Several of the candidate genes were up-regulated after infection with the fungus Colletotrichum lindemuthianum.

Reação de acessos tradicionais de feijão comum à Colletotrichum lindemuthianum

O feijoeiro comum é uma das culturas de elevada relevância socioeconômica para o Brasil. No entanto, as diferentes regiões de cultivo da cultura têm favorecido o surgimento de pragas e doenças, as quais afetam grandemente a produtividade de grãos. A antracnose do feijoeiro é altamente prejudicial à cultura, pois as perdas podem ser elevadas e/ou depreciar a qualidade do produto tornando-o menos atrativo ao comércio. Com isso, objetivou-se avaliar a detecção de acessos compatíveis com o C. lindemuthianum e a reação de resistência e suscetibilidade de 22 acessos tradicionais do feijoeiro comum às raças 73 e 89 de C. lindemuthianum. Na análise de sanidade foi utilizado o teste de incubação em rolo de papel de germinação, cujas sementes foram acondicionadas por 10 dias na BOD a uma temperatura de 20°C ± 2°. Na avaliação da reação de suscetibilidade e resistência ao patógeno utilizou-se ás raças 73 e 89 de C. lindemuthianum. O inóculo foi obtido a partir de culturas monospóricas das respectivas raças previamente preparadas. A suspensão de esporos foi ajustada a concentração aproximada de 1,2 x 106 esporos mL-1 de água destilada esterilizada. Os resultados da análise da detecção C. lindemuthianum demonstraram que em 81,2 % dos acessos não foram detectados lesões ou esporos do patógeno, que indicassem a contaminação dos acessos com o patógeno. A reação de suscetibilidade e resistência á raça 73 e 89 apresentou um índice de virulência de 70,58% e 12,5% para as respectivas raças dentre os acessos analisados.

New genomic regions for resistance to anthracnose (Colletotrichum lindemuthianum) through GBS-based genome-wide association study in common bean (Phaseolus vulgaris)

The most effective strategy to manage bean anthracnose (ANT), caused by Colletotrichum lindemuthianum, is the use of resistant cultivars. This study aimed to evaluate resistance reactions of common bean accessions to C. lindemuthianum races 2, 9 and 1545, and to perform genome-wide association study (GWAS). Hence, 89 accessions were phenotyped and genotyped through genotyping by sequencing (GBS). As a result, 48 accessions resistant to all evaluated races were identified. Moreover, single-nucleotide polymorphisms (SNP) significantly associated with resistance were identified in new regions of chromosomes Pv03, Pv05 and Pv06, and also at the beginning of Pv04 and end of Pv11, where other resistance genes have been previously found. In reference genome these regions contain model genes encoding resistance proteins as kinases, leucine-rich repeats, receptor-like protein, copper transport protein, pentatricopeptide repeats, calcium-dependent protein kinases, and ethylene-responsive transcription factors. The genomic regions associated to ANT resistance found in this study should be validated for further use in marker assisted selection and gene pyramiding. Together with new sources of ANT resistance our findings show promise for further crop improvement.

Response of Two Local Common Bean Ecotypes of “Fagioli di Sarconi” PGI (Phaseolus vulgaris L.) to Seed-Borne Pathogens and Environmental Change

Among foods protected by the European Union with the PGI (Protected Geographical Indication) mark, several ecotypes of “Fagioli di Sarconi” common beans, typical legumes of Basilicata Region, are included. This work aimed to conduct a survey of seed-borne pathogens isolated from “Ciuoto” and “Cannellino rosso”, ecotypes of “Fagioli di Sarconi” common beans, in two years and cultivation areas of the National Park of the Agri Valley, for identifying resistant and climatic changes well-adapted genotypes. Three validated methods were used for the seed-borne pathogens screening. Eighteen fungi were differently found for ecotype and year of observation by the washing test. Saprophyte contaminants pathogens isolated and detected by the blotter test were strongly reduced by 1% sodium hypochlorite treatment. Using the between paper test, specific for detecting Colletotrichum lindemuthianum, the presence of this pathogen for both ecotypes, years and cultivation areas, and also some bacteria were individuated. Therefore, area-, environment- and ecotype-dependent differences were revealed, probably also caused by a different polyphenolic content and thickness of integument of two ecotypes. This study represents a baseline information for further studies, development of forecasting models and management of seed-borne diseases associated with common beans.

Occurrence of anthracnose pathogen races and resistance genes in common bean across 30 years in Brazil

Anthracnose caused by Colletotrichum lindemuthianum is one of the most critical diseases in the common bean (Phaseolus vulgaris L.). The characterization and localization of pathogenic fungal races are essential for understanding pathogen population dynamics and recommending strategies to develop resistant cultivars. As resistant genotypes are the most economical and ecologically safe means of controlling plant diseases, there have been efforts to characterize resistance genes in common bean. Several studies using a system of 12 differential bean cultivars have been carried out to monitor anthracnose since 1991, reporting the constant appearance of new fungal races. C. lindemuthianum shows high virulence diversity. The objective of the present study was to review the relationship between C. lindemuthianum races and the common bean pathogenic processes involved in the risk of developing anthracnose disease. As a result, 89 races occurred in Brazil, wherein 73, 65, and 81 of C. lindemuthianum are the most frequent. Furthermore, we built a map with the anthracnose resistance loci, molecular markers, and their respective physical position. The accessibility to the genomes and sequencing technologies permits molecular markers for marker-assisted selection applied to anthracnose-resistant cultivars. This study could be used as a reference for future resistance mapping studies and as a guide for selecting resistance loci in breeding programs aiming to develop common bean cultivars with durable anthracnose resistance.