Genome-Wide Analysis and Evolution of the Pto-Like Protein Kinase (PLPK) Gene Family in Pepper

Abstract

The tomato Pto gene, which encodes a serine/threonine kinase (STK) domain-containing protein, confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato (Pst). In this study, in vivo recognition assays using PVX constructs showed that AvrPto was specifically recognized in the pepper genotypes. This AvrPto recognition caused a nonhost hypersensitive response (HR) and localization of the PVX::AvrPto fusion protein to inoculated pepper leaf tissues, which indicates the presence of a similar Pto recognition mechanism in pepper as in tomato. However, genome-wide analysis in pepper revealed no Pto clade corresponding to that in tomato, suggesting an alternative system for Pto recognition in pepper. Nevertheless, 25 Pto-like protein kinases (PLPKs) with a highly conserved STK domain have been identified in the pepper genome. For the majority of the amino acid sites in the STK domain of Ptos and PLPKs, nonsynonymous (dN) to synonymous (dS) nucleotide substitution ratios (ω) were less than one, suggesting that purifying selection played a predominant role in the evolutionary process. However, some amino acid sites were found to be subjected to episodic positive selection in the course of evolution of Pto homologs, and, thus, different evolutionary processes might have shaped the Pto gene family in plants. Based on RNA-seq data, PLPK genes and other Pto pathway genes, such as Prf, Pti1, Pti5, and Pti6 were expressed in all tested pepper genotypes. Therefore, the nonhost HR against Pst in pepper may be due to the recognition of the AvrPto effector by a PLPK homolog, and subsequent action of downstream components of the Pto signaling pathway. However, the possibility remains that the recognition of AvrPto in pepper plants may involve activities of other receptor like kinases (RLKs). The identification of the PLPKs in this study will serve as a foundation for further efforts to understand the roles of PLPKs in nonhost resistance.

Fig 1. Pepper seedlings inoculated with PVX vectors for expression of GFP, AvrPto and AvrPto^I96T.

A: Transient expression of AvrPto and AvrPto^I96T using a PVX-derived vector system in C. annuum ‘ECW’ (upper panels) and C. chinense ‘PI159234’ (lower panels). Plants were photographed at 14 dpi. Panels I, II: pPVX::GPF; Panels III, IV: pPVX::AvrPto; Panels V, VI: pPVX::AvrPto^I96T. B: Gel blot hybridization analysis of PVX RNA accumulation and the presence of AvrPto in uninoculated upper leaves of C. annuum ECW and C. chinense ‘PI159234’ infected with pPVX vector constructs. Total RNA was prepared from upper uninoculated leaves 14 dpi with the PVX constructs above or from mock-inoculated plants, blotted onto Hybond N⁺, and hybridized with a radiolabeled PVX-derived sequence (upper panels) and AvrPto (middle panels). Lane 1, mock-inoculated control; Lane 2, pPVX vector alone; Lane 3, pPVX::GFP; Lane 4, pPVX::AvrPto; and Lane 5, pPVX::AvrPto^I96T. The lower panel shows ethidium bromide-stained ribosomal RNA prior to blotting.

Fig 2. Phylogenetic analysis of tomato Pto paralogs and PLPKs.

A rooted phylogenetic tree was constructed using the predicted pepper PLPK sequences and full-length Pto proteins from tomato, N. benthamiana, potato, Arabidopsis and rice. The phylogenetic tree was constructed using the NJ method (1000 bootstrap replicates) as implemented in the MEGA 6.0 software. The name is indicated next to each subclass. Pto-like proteins from various plant species are classified into two main classes, Pto and PLPKs. PLPKs are further divided into eight subclasses (PLPK I-PLKPK VIII).

Fig 3. Phylogenetic analysis of pepper PLPKs.

A: A rooted phylogenetic tree was constructed using the predicted pepper PLPK sequences. The phylogenetic tree was constructed using the NJ method (1000 bootstrap replicates) as implemented in the MEGA 6.0 software. The name of each subclass is indicated. Phylogenetic analysis showed distinct clustering of PLPKs into eight subclasses (PLPK I–PLKPK VIII). B: Exon-intron structures of pepper PLPK genes. UTRs and exons are shown as blue and dark cyan boxes, respectively, and introns are shown as black lines. C: Structural and functional domains identified by the SMART program. Most pepper PLPKs contained a signal peptide region (red box), a malectin-like region, a transmembrane (TM) region (blue box), and a STK domain. PLPK18, which belongs to the PLPK V class, has an additional kinase domain at the C-terminus. PLPK7, which belongs to the PLPK VIII subclass, includes two stress-antifung domains at the N-terminus. In addition, low complexity regions (violet box) were present in several PLPKs.

Fig 4. Multiple sequence alignments of the STK region.

The STK domains were aligned using the Clustal Omega program, and the alignments were displayed in the "ClustalX" color mode available in JalView 2.8. The conserved domains I through XI are indicated in the figure. Autophophorylation sites are indicated with asterisks.

Fig 5. Expression profiles of PLPK genes from five different pepper accessions.

A heatmap was generated using log2 transformed RPKM expression values. The color bar at the top represents log2 expression values. The green, black and red colors represent high, medium, and low expression levels. A; PI260429, B; PI152225, C; PI201234, D; YCM334, E; Aji Dulce.