Fig 1.
Pepper seedlings inoculated with PVX vectors for expression of GFP, AvrPto and AvrPtoI96T.
A: Transient expression of AvrPto and AvrPtoI96T 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::AvrPtoI96T. 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::AvrPtoI96T. The lower panel shows ethidium bromide-stained ribosomal RNA prior to blotting.
Table 1.
Pto-like genes in pepper and other pepper genes related to Pto pathway.
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.
Table 2.
Positive selection analysis of the STK domain of Pto-like genes from various plants.
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.