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

Abstract

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.

Keywords: Candidate gene expression; Phaseolus vulgaris; Plant immunity; Pseudomonas syringae pv. phaseolicola; Transcriptome.

Fig. 1

Mapping of common bean genes modulated by Pseudomonas syringae pv. phaseolicola. Distribution of differentially expressed genes (DEGs) throughout the P. vulgaris genome. DEGs (total of 632) mapped to all 11 P. vulgaris chromosomes and scaffolds (genome version v2.1; https://phytozome.jgi.doe.gov/). The number on top of the bars indicates the total DEGs mapped to each genome region

Fig. 2

Protein domain enrichment analysis. a List of enriched protein domains (Holm–Bonferroni; > 5 genes and p < 0.01) identified among DEGs according to InterPro codes (http://www.ebi.ac.uk/interpro). b Venn diagram showing DEGs that encode leucine-rich repeat (LRR) and/or malectin domains. c Heatmap of expression levels (Z-ratio) of the seven DEG containing both malectin-like and LRR domains and their putative Arabidopsis orthologs (E value = 0.0, Identity > 45%)

Fig. 3

Chromosome location of the 38 common bean genes encoding malectin-like and/or LRR domains regulated by Pph. The numbers on the left are the physical positions of each gene in the common bean chromosomes (in Mb). Letters “a” and “b” indicate the beginning and end of the chromosome, respectively. Genes tagged with green, blue, or orange code for LRR domain, malectin-like domain, or proteins with both LRR and malectin-like domains, respectively. The black rectangles on the left of the chromosomes indicate the physical position of the previously mapped Pph resistance QTLs (Miklas et al. ; Trabanco et al. ; Tock et al. 2017). Brown arrows point to genes that had their expression assessed via RT-qPCR. The gene Phvul.008G030800 (*) was previously described (Oblessuc et al. 2015). The map was created using MapChart software (Voorrips 2002)

Fig. 4

Phylogenetic analysis of DEGs encoding malectin and/or LRR domains. Complete predicted proteins were aligned using the ClustalW algorithm in MEGAX. Phylogenetic clustering was accomplished using the neighbor-joining method with 1000 bootstraps. The number in front of the gene ID is the Z-ratio differential expression, wherein red and blue fonts indicate downregulation and upregulation, respectively, upon bacterium infection. The putative Arabidopsis thaliana ortholog (E value < 10^–31) for each P. vulgaris protein is listed on the right. Genes tagged with green, blue, and orange squares code for LRR domain, malectin-like domain, and proteins with both LRR and malectin-like domains, respectively

Fig. 5

Pseudomonas syringae pv. phaseolicola growth in four common bean genotypes over 7 days. Data are presented as means ± standard error (n = 3). Significant differences in bacterial populations between 0 and 7 day post-inoculation (dpi) were measured via paired Student’s t test (α = 0.05) and are indicated with an asterisk (*). Bacterial cells were not recovered from the resistant genotypes Jalo EEP and AND 227 at 7 dpi; thus, statistical tests could not be performed for these genotypes

Fig. 6

Relative expression of select genes in common bean genotypes inoculated with P. syringae pv. phaseolicola. Gene expression at 12 hpi was determined by RT-qPCR. Gene expression data were analyzed using the 2^−ΔΔCq method, log₂ transformed, and are presented as means ± standard error (n = 3). Significant differences in the expression of each gene in bacterium-inoculated plants compared to mock control were determined via paired Student’s t test (α = 0.05) and are signified by an asterisk (*). ns non-significant

Fig. 7

Common bean candidate genes and their encoded protein domains that promote defense against Pph. Common bean malectin-domain protein FER-like (Oblessuc et al. 2015), together with the malectin–LRR putative homologs of XLG2 and IOS1 (mapped at the same location as HB5.1; Tock et al. 2017), would work together with BAK1-like and other LRR domain proteins co-localized with the major QTL Pse-6/HB4.2 (Tock et al. 2017) to promote plant defenses against Pph. Genes in green letters had their expression verified by RT-qPCR