Pseudomonas phaseolicola preferentially modulates genes encoding leucine-rich repeat and malectin domains in the bean landrace G2333.

MAIN CONCLUSION: Candidate resistance genes encoding malectin-like and LRR domains mapped to halo blight resistance loci throughout the common bean genome are co-expressed to fight a range of Pph races. Common bean (Phaseolus vulgaris L.) is an important crop both as a source of protein and other nutrients for human nutrition and as a nitrogen fixer that benefits sustainable agriculture. This crop is affected by halo blight disease, caused by the bacterium Pseudomonas syringae pv. phaseolicola (Pph), which can lead to 45% yield losses. Common bean resistance to Pph is conferred by six loci (Pse-1 to Pse-6) and minor-effect quantitative trait loci (QTLs); however, information is lacking on the molecular mechanisms implicated in this resistance. Here, we describe an in-depth RNA-sequencing (RNA-seq) analysis of the tolerant G2333 bean line in response to the Pph strain NPS3121. We identified 275 upregulated and 357 downregulated common bean genes in response to Pph infection. These differentially expressed genes were mapped to all 11 chromosomes of P. vulgaris. The upregulated genes were primarily components of plant immune responses and negative regulation of photosynthesis, with enrichment for leucine-rich repeat (LRRs) and/or malectin-like carbohydrate-binding domains. Interestingly, LRRs and malectin genes mapped to the same location as previously identified Pph resistance loci or QTLs. For instance, the major loci Pse-6/HB4.2 involved in broad-resistance to many Pph races co-located with induced LRR-encoding genes on Pv04. These findings indicate a coordinated modulation of genes involved in pathogen perception and signal transduction. In addition, the results further support these LRR/malectin loci as resistance genes in response to halo blight. Thus, these genes are potential targets for future genetic manipulation, enabling the introduction of resistance to Pph into elite cultivars of common bean.

Genome editing for resistance against plant pests and pathogens.

The conventional breeding of crops struggles to keep up with increasing food needs and ever-adapting pests and pathogens. Global climate changes have imposed another layer of complexity to biological systems, increasing the challenge to obtain improved crop cultivars. These dictate the development and application of novel technologies, like genome editing (GE), that assist targeted and fast breeding programs in crops, with enhanced resistance to pests and pathogens. GE does not require crossings, hence avoiding the introduction of undesirable traits through linkage in elite varieties, speeding up the whole breeding process. Additionally, GE technologies can improve plant protection by directly targeting plant susceptibility (S) genes or virulence factors of pests and pathogens, either through the direct edition of the pest genome or by adding the GE machinery to the plant genome or to microorganisms functioning as biocontrol agents (BCAs). Over the years, GE technology has been continuously evolving and more so with the development of CRISPR/Cas. Here we review the latest advancements of GE to improve plant protection, focusing on CRISPR/Cas-based genome edition of crops and pests and pathogens. We discuss how other technologies, such as host-induced gene silencing (HIGS) and the use of BCAs could benefit from CRISPR/Cas to accelerate the development of green strategies to promote a sustainable agriculture in the future.

A Simple Assay to Assess Salmonella enterica Persistence in Lettuce Leaves After Low Inoculation Dose.

Salmonella enterica is an enterobacterium associated with numerous foodborne illnesses worldwide. Leafy greens have been a common vehicle for disease outbreaks caused by S. enterica. This human pathogen can be introduced into crop fields and potentially contaminate fresh produce. Several studies have shown that S. enterica can survive for long periods in the plant tissues. Often, S. enterica population does not reach high titers in leaves; however, it is still relevant for food safety due to the low infective dose of the pathogen. Thus, laboratory procedures to study the survival of S. enterica in fresh vegetables should be adjusted accordingly. Here, we describe a protocol to assess the population dynamics of S. enterica serovar Typhimurium 14028s in the leaf apoplast of three cultivars of lettuce (Lactuca sativa L.). By comparing a range of inoculum concentrations, we showed that vacuum infiltration of a bacterium inoculum level in the range of 3.4 Log CFU ml-1 (with a recovery of approximately 170 cells per gram of fresh leaves 2 h post inoculation) allows for a robust assessment of bacterial persistence in three lettuce cultivars using serial dilution plating and qPCR methods. We anticipate that this method can be applied to other leaf-human pathogen combinations in an attempt to standardize the procedure for future efforts to screen for plant phenotypic variability, which is useful for breeding programs.

Novel molecular components involved in callose-mediated Arabidopsis defense against Salmonella enterica and Escherichia coli O157:H7.

BACKGROUND: Food contamination with Salmonella enterica and enterohemorrhagic Escherichia coli is among the leading causes of foodborne illnesses worldwide and crop plants are associated with > 50% of the disease outbreaks. However, the mechanisms underlying the interaction of these human pathogens with plants remain elusive. In this study, we have explored plant resistance mechanisms against these enterobacteria and the plant pathogen Pseudomonas syringae pv. tomato (Pst) DC3118, as an opportunity to improve food safety. RESULTS: We found that S. enterica serovar Typhimurium (STm) transcriptionally modulates stress responses in Arabidopsis leaves, including induction of two hallmark processes of plant defense: ROS burst and cell wall modifications. Analyses of plants with a mutation in the potentially STm-induced gene EXO70H4 revealed that its encoded protein is required for stomatal defense against STm and E. coli O157:H7, but not against Pst DC3118. In the apoplast however, EXO70H4 is required for defense against STm and Pst DC3118, but not against E. coli O157:H7. Moreover, EXO70H4 is required for callose deposition, but had no function in ROS burst, triggered by all three bacteria. The salicylic acid (SA) signaling and biosynthesis proteins NPR1 and ICS1, respectively, were involved in stomatal and apoplastic defense, as well as callose deposition, against human and plant pathogens. CONCLUSIONS: The results show that EXO70H4 is involved in stomatal and apoplastic defenses in Arabidopsis and suggest that EXO70H4-mediated defense play a distinct role in guard cells and leaf mesophyll cells in a bacteria-dependent manner. Nonetheless, EXO70H4 contributes to callose deposition in response to both human and plant pathogens. NPR1 and ICS1, two proteins involved in the SA signaling pathway, are important to inhibit leaf internalization and apoplastic persistence of enterobacteria and proliferation of phytopathogens. These findings highlight the existence of unique and shared plant genetic components to fight off diverse bacterial pathogens providing specific targets for the prevention of foodborne diseases.

The common bean COK-4 and the Arabidopsis FER kinase domain share similar functions in plant growth and defence.

Receptor-like kinases are membrane proteins that can be shared by diverse signalling pathways. Among them, the Arabidopsis thaliana FERONIA (FER) plays a role in the balance between distinct signals to control growth and defence. We have found that COK-4, a putative kinase encoded in the common bean anthracnose resistance locus Co-4, which is transcriptionally regulated during the immune response, is highly similar to the kinase domain of FER. To assess whether COK-4 is a functional orthologue of FER, we expressed COK-4 in the wild-type Col-0 and the fer-5 mutant of Arabidopsis and evaluated FER-associated traits. We observed that fer-5 plants show an enhanced apoplastic and stomatal defence against Pseudomonas syringae. In addition, the fer-5 mutant shows reduced biomass, smaller guard cell size, greater number of stomata per leaf area, fewer leaves, faster transition to reproductive stage and lower seed weight per plant than the wild-type Col-0. Except for the stomatal complex length and number of stomata, COK-4 expression in fer-5 lines partially or completely rescued both defence and developmental defects of fer-5 to the wild-type level. Notably, COK-4 may have an additive effect to FER, as the expression of COK-4 in Col-0 resulted in enhanced defence and growth phenotypes in comparison with wild-type Col-0 plants. Altogether, these findings indicate that the common bean COK-4 shares at least some of the multiple functions of the Arabidopsis FER kinase domain, acting in both the induction of plant growth and regulation of plant defence.

Linkage and mapping of quantitative trait loci associated with angular leaf spot and powdery mildew resistance in common beans

Abstract Angular leaf spot (ALS) and powdery mildew (PWM) are two important fungi diseases causing significant yield losses in common beans. In this study, a new genetic linkage map was constructed using single sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), in a segregating population derived from the AND 277 x SEA 5 cross, with 105 recombinant inbred lines. Phenotypic evaluations were performed in the greenhouse to identify quantitative trait loci (QTLs) associated with resistance by means of the composite interval mapping analysis. Four QTLs were identified for ALS resistance. The QTL ALS11AS, linked on the SNP BAR 5054, mapped on chromosome Pv11, showed the greatest effect (R2 = 26.5%) on ALS phenotypic variance. For PWM resistance, two QTLs were detected, PWM2AS and PWM11AS, on Pv2 and Pv11, explaining 7% and 66% of the phenotypic variation, respectively. Both QTLs on Pv11 were mapped on the same genomic region, suggesting that it is a pleiotropic region. The present study resulted in the identification of new markers closely linked to ALS and PWM QTLs, which can be used for marker-assisted selection, fine mapping and positional cloning.

Linkage and mapping of quantitative trait loci associated with angular leaf spot and powdery mildew resistance in common beans.

Angular leaf spot (ALS) and powdery mildew (PWM) are two important fungi diseases causing significant yield losses in common beans. In this study, a new genetic linkage map was constructed using single sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), in a segregating population derived from the AND 277 x SEA 5 cross, with 105 recombinant inbred lines. Phenotypic evaluations were performed in the greenhouse to identify quantitative trait loci (QTLs) associated with resistance by means of the composite interval mapping analysis. Four QTLs were identified for ALS resistance. The QTL ALS11AS, linked on the SNP BAR 5054, mapped on chromosome Pv11, showed the greatest effect (R2 = 26.5%) on ALS phenotypic variance. For PWM resistance, two QTLs were detected, PWM2AS and PWM11AS, on Pv2 and Pv11, explaining 7% and 66% of the phenotypic variation, respectively. Both QTLs on Pv11 were mapped on the same genomic region, suggesting that it is a pleiotropic region. The present study resulted in the identification of new markers closely linked to ALS and PWM QTLs, which can be used for marker-assisted selection, fine mapping and positional cloning.

Genome-Wide Association Studies of Anthracnose and Angular Leaf Spot Resistance in Common Bean (Phaseolus vulgaris L.).

The common bean (Phaseolus vulgaris L.) is the world's most important legume for human consumption. Anthracnose (ANT; Colletotrichum lindemuthianum) and angular leaf spot (ALS; Pseudocercospora griseola) are complex diseases that cause major yield losses in common bean. Depending on the cultivar and environmental conditions, anthracnose and angular leaf spot infections can reduce crop yield drastically. This study aimed to estimate linkage disequilibrium levels and identify quantitative resistance loci (QRL) controlling resistance to both ANT and ALS diseases of 180 accessions of common bean using genome-wide association analysis. A randomized complete block design with four replicates was performed for the ANT and ALS experiments, with four plants per genotype in each replicate. Association mapping analyses were performed for ANT and ALS using a mixed linear model approach implemented in TASSEL. A total of 17 and 11 significant statistically associations involving SSRs were detected for ANT and ALS resistance loci, respectively. Using SNPs, 21 and 17 significant statistically associations were obtained for ANT and angular ALS, respectively, providing more associations with this marker. The SSR-IAC167 and PvM95 markers, both located on chromosome Pv03, and the SNP scaffold00021_89379, were associated with both diseases. The other markers were distributed across the entire common bean genome, with chromosomes Pv03 and Pv08 showing the greatest number of loci associated with ANT resistance. The chromosome Pv04 was the most saturated one, with six markers associated with ALS resistance. The telomeric region of this chromosome showed four markers located between approximately 2.5 Mb and 4.4 Mb. Our results demonstrate the great potential of genome-wide association studies to identify QRLs related to ANT and ALS in common bean. The results indicate a quantitative and complex inheritance pattern for both diseases in common bean. Our findings will contribute to more effective screening of elite germplasm to find resistance alleles for marker-assisted selection in breeding programs.

The Co-4 locus on chromosome Pv08 contains a unique cluster of 18 COK-4 genes and is regulated by immune response in common bean.

KEY MESSAGE: The common bean locus Co - 4, traditionally referred to as an anthracnose-resistant gene, contains a cluster of predicted receptor-like kinases (COK-4 and CrRLK1-like), and at least two of these kinases are co-regulated with the plant's basal immunity. Genetic resistance to anthracnose, caused by the fungus Colletotrichum lindemuthianum (Sacc. and Magnus) Briosi and Cavara, is conferred by major loci throughout the Phaseolus vulgaris genome, named Co. The complex Co-4 locus was previously reported to have several copies of the COK-4 gene that is predicted to code for a receptor-like kinase (RLK). In general, plant RLKs are involved in pathogen perception and signal transduction; however, the molecular function of COK-4 remains elusive. Using newly identified molecular markers (PvTA25 and PvSNPCOK-4), the SAS13 marker, COK-4 sequences and phylogeny, and the recently released bean genome sequence, we determined the most probable boundaries of the Co-4 locus: a 325-Kbp region on chromosome Pv08. Out of the 49 predicted transcripts in that region, 24 encode for putative RLKs (including 18 COK-4 copies) with high similarity to members of the Catharanthus roseus RLK1-like (CrRLK1L) protein family from different plant species, including the well-described FERONIA (FER) and ANXUR. We also determined that two RLK-coding genes in the Co-4 locus (COK-4-3 and FER-like) are transcriptionally regulated when bean plants are challenged with the flg22 peptide, a commonly used elicitor of plant immunity, or the bacterium Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight. While COK-4-3 is activated during immune response, FER-like is downregulated suggesting that these genes could play a role in plant responses to biotic stress. These results highlight the importance of dissecting the regulation and molecular function of individual genes within each locus, traditionally referred to as resistance gene based on genetic segregation analysis.

Increasing the density of markers around a major QTL controlling resistance to angular leaf spot in common bean.

Angular leaf spot (ALS) causes major yield losses in the common bean (Phaseolus vulgaris L.), an important protein source in the human diet. This study describes the saturation around a major quantitative trait locus (QTL) region, ALS10.1, controlling resistance to ALS located on linkage group Pv10 and explores the genomic context of this region using available data from the P. vulgaris genome sequence. DArT-derived markers (STS-DArT) selected by bulk segregant analysis and SCAR and SSR markers were used to increase the resolution of the QTL, reducing the confidence interval of ALS10.1 from 13.4 to 3.0 cM. The position of the SSR ATA220 coincided with the maximum LOD score of the QTL. Moreover, a new QTL (ALS10.2(UC)) was identified at the end of the same linkage group. Sequence analysis using the P. vulgaris genome located ten SSRs and seven STS-DArT on chromosome 10 (Pv10). Coincident linkage and genome positions of five markers enabled the definition of a core region for ALS10.1 spanning 5.3 Mb. These markers are linked to putative genes related to disease resistance such as glycosyl transferase, ankyrin repeat-containing, phospholipase, and squamosa-promoter binding protein. Synteny analysis between ALS10.1 markers and the genome of soybean suggested a dynamic evolution of this locus in the common bean. The present study resulted in the identification of new candidate genes and markers closely linked to a major ALS disease resistance QTL, which can be used in marker-assisted selection, fine mapping and positional QTL cloning.

Dissecting Phaseolus vulgaris innate immune system against Colletotrichum lindemuthianum infection.

BACKGROUND: The genus Colletotrichum is one of the most economically important plant pathogens, causing anthracnose on a wide range of crops including common beans (Phaseolus vulgaris L.). Crop yield can be dramatically decreased depending on the plant cultivar used and the environmental conditions. This study aimed to identify potential genetic components of the bean immune system to provide environmentally friendly control measures against this fungus. METHODOLOGY AND PRINCIPAL FINDINGS: As the common bean is not amenable to reverse genetics to explore functionality and its genome is not fully curated, we used putative Arabidopsis orthologs of bean expressed sequence tag (EST) to perform bioinformatic analysis and experimental validation of gene expression to identify common bean genes regulated during the incompatible interaction with C. lindemuthianum. Similar to model pathosystems, Gene Ontology (GO) analysis indicated that hormone biosynthesis and signaling in common beans seem to be modulated by fungus infection. For instance, cytokinin and ethylene responses were up-regulated and jasmonic acid, gibberellin, and abscisic acid responses were down-regulated, indicating that these hormones may play a central role in this pathosystem. Importantly, we have identified putative bean gene orthologs of Arabidopsis genes involved in the plant immune system. Based on experimental validation of gene expression, we propose that hypersensitive reaction as part of effector-triggered immunity may operate, at least in part, by down-regulating genes, such as FLS2-like and MKK5-like, putative orthologs of the Arabidopsis genes involved in pathogen perception and downstream signaling. CONCLUSIONS/SIGNIFICANCE: We have identified specific bean genes and uncovered metabolic processes and pathways that may be involved in the immune response against pathogens. Our transcriptome database is a rich resource for mining novel defense-related genes, which enabled us to develop a model of the molecular components of the bean innate immune system regulated upon pathogen attack.

Mapping of angular leaf spot resistance QTL in common bean (Phaseolus vulgaris L.) under different environments.

BACKGROUND: Common bean (Phaseolus vulgaris L.) is the most important grain legume for human diet worldwide and the angular leaf spot (ALS) is one of the most devastating diseases of this crop, leading to yield losses as high as 80%. In an attempt to breed resistant cultivars, it is important to first understand the inheritance mode of resistance and to develop tools that could be used in assisted breeding. Therefore, the aim of this study was to identify quantitative trait loci (QTL) controlling resistance to ALS under natural infection conditions in the field and under inoculated conditions in the greenhouse. RESULTS: QTL analyses were made using phenotypic data from 346 recombinant inbreed lines from the IAC-UNAxCAL 143 cross, gathered in three experiments, two of which were conducted in the field in different seasons and one in the greenhouse. Joint composite interval mapping analysis of QTLxenvironment interaction was performed. In all, seven QTLs were mapped on five linkage groups. Most of them, with the exception of two, were significant in all experiments. Among these, ALS10.1DG,UC presented major effects (R2 between 16%-22%). This QTL was found linked to the GATS11b marker of linkage group B10, which was consistently amplified across a set of common bean lines and was associated with the resistance. Four new QTLs were identified. Between them the ALS5.2 showed an important effect (9.4%) under inoculated conditions in the greenhouse. ALS4.2 was another major QTL, under natural infection in the field, explaining 10.8% of the variability for resistance reaction. The other QTLs showed minor effects on resistance. CONCLUSIONS: The results indicated a quantitative inheritance pattern of ALS resistance in the common bean line CAL 143. QTL x environment interactions were observed. Moreover, the major QTL identified on linkage group B10 could be important for bean breeding, as it was stable in all the environments. Thereby, the GATS11b marker is a potential tool for marker assisted selection for ALS resistance.