Identification and Expression Profiling of WRKY Family Genes in Sugarcane in Response to Bacterial Pathogen Infection and Nitrogen Implantation Dosage

Abstract

WRKY transcription factors (TFs) are essential players in different signaling cascades and regulatory networks involved in defense responses to various stressors. This study systematically analyzed and characterized WRKY family genes in the Saccharum spp. hybrid R570 and their expression in two sugarcane cultivars LCP85-384 (resistant to leaf scald) and ROC20 (susceptible to leaf scald) in response to bacterial pathogen infection and nitrogen implantation dosage. A total of 53 ShWRKY genes with 66 alleles were systematically identified in R570 based on the query sequence SsWRKY in S. spontaneum AP85-441. All ShRWKY alleles were further classified into four groups with 11 (16.7%) genes in group I, 36 (54.5%) genes in group II, 18 (27.3%) genes in group III, and 1 (1.5%) gene in group IV. Among them, 4 and 11 ShWRKY gene pairs displayed tandem and segmental duplication events, respectively. The ShWRKY genes exhibited conserved DNA-binding domains, which were accompanied by variations in introns, exons, and motifs. RT-qPCR analysis of two sugarcane cultivars triggered by Xanthomonas albilineans (Xa) revealed that four genes, ShWRKY13-2/39-1/49-3/125-3, exhibited significant upregulation in leaf scald-resistant LCP85-384. These WRKY genes were downregulated or unchanged in ROC20 at 24-72 h post-inoculation, suggesting that they play an important role in defense responses to Xa infection. Most of the 12 tested ShWRKYs, ShWRKY22-1/49-3/52-1 in particular, functioned as negative regulators in the two cultivars in response to a range of nitrogen (N) implantation doses. A total of 11 ShWRKY proteins were predicted to interact with each other. ShWRKY43 and ShWRKY49-3 are predicted to play core roles in the interaction network, as indicated by their interaction with six other ShWRKY proteins. Our results provide important candidate gene resources for the genetic improvement of sugarcane and lay the foundation for further functional characterization of ShWRKY genes in response to coupling effects of Xa infection and different N levels.

Keywords: Saccharum spp.; WRKY transcription factors; Xanthomonas albilineans; gene regulation; nitrogen dosage.

FIGURE 1

Phylogenetic tree of ShWRKYs. The unrooted NJ tree was constructed based on the WRKY domains from Saccharum spp. hybrid R570 using MEGA7.0 with bootstraps of 1,000 replicates. Group and subgroup names are on the outer ring and are depicted in different colors. Black stars, red boxes, blue triangles, and green circles represent different ShWRKY groups I, II, III, and IV, respectively.

FIGURE 2

(A) Locations and lengths of ShWRKY exons and introns which are shown as filled yellow bars and thin gray single lines, respectively. UTRs are represented by dark blue bars at the ends of the lines. Gene structures were drawn using the GSDS online database. (B) Conserved motif analysis of ShWRKYs. All motifs were identified using the MEME online database and visualized with Tbtools software. A total of ten predicted motifs are represented by different colored boxes and motif sizes can be estimated using the scale at the bottom.

FIGURE 3

Heatmap of cis-regulatory elements in the ShWRKY genes. Blue: Present, Yellow: Absent.

FIGURE 4

Chromosomal distribution of ShWRKY genes in Saccharum spp. hybrid R570. Ribbon links (Red lines) indicate segmental duplication events between genes. Chromosome numbers are indicated inside the yellow segments. Chr01 to Chr10 are currently based on Sorghum bicolor genome scaffolds (https://phytozome-next.jgi.doe.gov/info/SbicolorSC187_v1_1). The Saccharum spp. R570 gff3 file contains these 10 chromosomes and all BAC clones that could not be placed. The gene names on each chromosome are indicated in the outer circle.

FIGURE 5

Expression profiling of twelve ShWRKY genes at different times post-Xa inoculation in LCP85-384 (black gars) and ROC20 (gray bars) cultivars. Values for relative expression levels are means ± standard errors. Means having the same letters above the vertical bars are not significantly different at a 5% level of probability.

FIGURE 6

Expression profiling of twelve ShWRKY genes at different times after nitrogen implantation dosage in LCP85-384 and ROC20 cultivars. Values are means ± standard errors. Means having the same letters above the vertical bars are not significantly different at a 5% level of probability.

FIGURE 7

Predicted protein-protein interactions of ShWRKYs according to their orthologs in A. thaliana. Only those pairs with >30% sequence identity between ShWRKYs and AtWRKYs and an interaction score >0.8 are shown in the network. Line and node colors indicate different kinds and degrees of interactions, respectively. Ribbon diagrams are shown in each node.