Genome-wide identification of TaeGRASs responsive to biotic stresses and functional analysis of TaeSCL6 in wheat resistance to powdery mildew

Abstract

Background: Powdery mildew is a devastating fungal disease that poses a significant threat to wheat yield and quality worldwide. Identifying resistance genes is highly advantageous for the molecular breeding of resistant cultivars. GRAS proteins are important transcription factors that regulate plant development and stress responses. Nonetheless, their roles in wheat-pathogen interactions remain poorly understood.

Results: In this study, we used bioinformatics tools to identify and analyze wheat GRAS family genes responsive to biotic stresses and elucidated the function of TaeSCL6 within this family. A total of 179 GRAS genes in wheat were unevenly distributed on 7 chromosomes, and classified into 12 subfamilies based on phylogenetic relationship analysis. Gene duplication analysis revealed 13 pairs of tandem repeats and 142 pairs of segmental duplications, which may account for the rapid expansion of the wheat GRAS family. Expression pattern analysis revealed that 75% of the expressed TaeGRAS genes are responsive to biotic stresses. Few studies have focused on the roles of HAM subfamily genes. Consequently, we concentrated our analysis on the members of the HAM subfamily. Fourteen motifs were identified in the HAM family proteins from both Triticeae species and Arabidopsis, indicating that these motifs were highly conserved during evolution. Promoter analysis indicated that the promoters of HAM genes contain several cis-regulatory elements associated with hormone response, stress response, light response, and growth and development. Both qRT-PCR and RNA-seq data analyses demonstrated that TaeSCL6 responds to Blumeria graminis infection. Therefore, we investigated the role of TaeSCL6 in regulating wheat resistance via RNA interference and barley stripe mosaic virus induced gene silencing. Wheat plants with silenced TaeSCL6 exhibited increased susceptibility to powdery mildew.

Conclusions: In summary, this study not only validates the positive role of TaeSCL6 in wheat resistance to powdery mildew, but also provides candidate gene resources for future breeding of disease-resistance wheat cultivars.

Keywords: GRAS; Powdery mildew; RNA interference; TaeSCL6; Virus-induced gene silencing; Wheat.

Fig. 1

Phylogenetic relationship analysis of GRAS proteins from Triticeae species and A. thaliana. The phylogenetic tree was built using the Maximum Likelihood method with 1,000 bootstrap replicates by TBtools. The diverse subfamilies of GRAS proteins were marked with different colors. The GRAS proteins are from T. aestivum (Traes), T. urartu (Tu), Ae. tauschii (AET), H. vulgare (HORVU), T. dicoccoides (TRID) and A. thaliana (AT), which are denoted by red stars, orange squares, green triangles, yellow stars, purple rectangles and blue circles, respectively

Fig. 2

Chromosomal distribution of TaeGRAS genes

Fig. 3

Analysis of duplication events of GRAS genes in wheat. The gray lines in the background represent the segmental duplications in the whole wheat genome. Red lines represent GRAS gene pairs with segmental duplications. The first ring from the outside indicates the chromosomal localization of 176 putative GRAS genes in wheat. The second and third rings from outside represent the density of genes on the chromosomes

Fig. 4

Analysis of the synteny of GRAS genes among five Triticeae species and A. thaliana. The gray lines between the genomes present all the synteny blocks, and red lines between the genomes indicate the synteny between the genes

Fig. 5

Analysis of conserved motifs and domains of HAM proteins in Triticeae species. (A) An unrooted phylogenetic tree of HAM proteins was constructed under the Maximum Likelihood method with 1000 bootstrap replicates using TBtools software. The red font indicates the three copies of TaeSCL6 in subgenomes A, B and D. (B) Conserved HAM protein motifs performed by MEME. The colored boxes refer to the motifs. (C) Domain analyses of HAM proteins were performed under the Gene Structure Display Server 2.0 program. The domains are displayed in different colored boxes

Fig. 6

Cis-acting elements in wheat HAM gene promoters. The color scale bar and the number represent the frequency of these cis-acting elements in the promoter

Fig. 7

Heatmap of the expression profiles of wheat GRAS genes under different stresses. The color scale bar represents the expression values (in log2-based tags per million values) of the genes, and the values in square frames represent the tags per million values. The phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrap replicates by TBtools. Bgt (E09), wheat variety N9134 infected with the race E09 of Blumeria graminis f. sp. Tritici (E09); Pst, wheat variety N9134 infected with the race CYR31 of Puccinia striiformis f. sp. tritici; Control, Disease-resistant wheat varieties N9134 without infection by Bgt and Pst; Water, wheat varieties Chinese Spring sprayed with water; Chitin, wheat variety Chinese Spring sprayed with chitin solution; Flag22, wheat variety Chinese Spring treated with bacteria flagellar protein flagellin 22 (Flag22)-derived peptides

Fig. 8

Functional analysis of TaeSCL6. (A) Relative expression levels of TaeSCL6 in plants infected with BSMV: TaeSCL6. BSMV:γ represents wheat cultivar Bainong207 plants infected with BSMV:γ, and BSMV:1, BSMV:2 and BSMV:3 represent different plants infected with BSMV: TaeSCL6. (B) Phenotypes of detached leaves from BSMV: TaeSCL6-infected plants and the control BSMV:γ-infected plants inoculated with Bgt. (C) Relative expression levels of TaeSCL6 in RNAi-TaeSCL6 transgenic plants. (D) The phenotypes of detached leaves from RNAi-TaeSCL6 transgenic plants and the control wheat cultivar AK58 inoculated with Bgt. CK represents wild-type plants AK58, and R1, R2, R3, and R4 represent different transgenic lines. (E) The phenotype of hyphal development in RNAi-TaeSCL6 transgenic plants. Sumai3 is used as a control, highly susceptible to powdery mildew. Error bars represent the standard deviations from three technical replicates. **** represents significant differences at the level of P < 0.001 using Tukey’s multiple comparisons test