Identification and Expression Analysis of NAC Transcription Factors Related to Rust Resistance in Foxtail Millet

Abstract

Foxtail millet (Setaria italica), a vital cereal crop in China, serves as both a staple food and forage source but is threatened by rust disease caused by Uromyces setariae-italicae (Usi), leading to severe yield and quality losses. The NAM, ATAF1/2, and CUC2 (NAC) transcription factor family represents one of the largest plant-specific regulatory gene families, playing pivotal roles in development and stress responses. However, the functional relevance of NAC genes in foxtail millet's defense against this pathogen remains unexplored. Here, we systematically analyzed 33 SiNAC genes from the foxtail millet genome. Phylogenetic analysis classified these genes into 11 subgroups, while chromosomal mapping localized them to nine chromosomes unevenly. Promoter analysis identified stress- and plant hormone-related cis-elements, suggesting functional diversity. Expression profiling analysis showed that most SiNAC genes exhibit tissue-specific expression patterns. Quantitative real-time PCR (qRT-PCR) results indicated that 30 genes responded to Usi infection, with 17 showing a strong association with rust resistance. Three resistance-associated genes demonstrated transactivation activity and nuclear localization, indicating their regulatory function in defense responses. This study provides both mechanistic insights into SiNAC-mediated rust resistance and potential targets for molecular breeding in foxtail millet.

Keywords: NAC; Setaria italica; Uromyces setariae-italicae; qRT-PCR; rust disease resistance; transcription factor.

Figure 1

Phylogenetic tree of NAC genes in Setaria italica, Oryza sativa, and Arabidopsis thaliana.

Figure 2

Chromosomal distribution of 33 SiNAC genes in Setaria italica. The left-hand scale denotes the genomic length in megabases (Mb).

Figure 3

Prediction of cis-acting elements in the promoters of 33 SiNAC genes. (A) Phylogenetic tree of SiNACs. The phylogenetic relationships among homologous proteins were reconstructed using MEGA5 software, employing the neighbor-joining algorithm with bootstrap validation (n = 1000 replicates) to assess topological robustness. (B) Cis-acting element structures in promoter regions of SiNACs. Different colors represent 13 kinds of 196 functional modules and unknown genome sequence.

Figure 4

Expression profiles of SiNAC genes across different growth stages and tissues. Expression levels are represented by a heatmap with color-coded Z-Scores (scale shown). Developmental stages: BT, booting stage; SD, seedling stage.

Figure 5

Differential expression patterns of 33 SiNAC genes in resistance responses. Relative expression level of SiNACs was calculated by the 2^−∆∆CT method. (A) SiNAC genes exhibiting significant upregulation. (B) SiNAC genes exhibiting significant downregulation. (C) Stable expression profiles of SiNAC genes. (D) Varied expression patterns of SiNAC genes. The vertical lines on the top of bars correspond to the standard error of the respective mean estimates.

Figure 6

Leaf phenotypes of YG1 and SLX at 14 days post-inoculation with Usi 93-5. The leaves of YG1 (Left) and SLX (right) foxtail millet plants were used to inoculate Usi 93-5, respectively.

Figure 7

Similar expression patterns of 13 SiNAC genes in resistant and susceptible responses. Relative expression level of SiNACs was calculated by the 2^−∆∆CT method. (A) Upregulation of SiNAC genes in both resistance and susceptible responses. (B) Downregulation of SiNAC genes in both resistance and susceptible responses. The vertical lines on the top of bars correspond to the standard error of the respective mean estimates.

Figure 8

Differential expression patterns of 17 SiNAC genes both in resistant and susceptible responses. Relative expression level of SiNACs was calculated by the 2^−∆∆CT method. (A) 9 SiNAC genes were upregulated at some or all time points during the incompatible interaction, while their expression was downregulated in the compatible interaction. (B) 4 SiNAC genes were specifically regulated during the incompatible interaction, with changes in transcript levels ranging from induction to repression, while no notable changes occurred in the compatible interaction. (C) 4 SiNAC genes displayed distinct changes in expression dynamics at different time points in both interactions. The vertical lines on the top of bars correspond to the standard error of the respective mean estimates.

Figure 9

Subcellular localization and transcriptional activation analysis of SiNAC063, SiNAC070, and SiNAC118 protein. (A) Fluorescence observation of SiNAC063, SiNAC070, and SiNAC118 protein. The empty p1104-YFP vector was used as a positive control. The mCherry fused with the nuclear localization signal was used as a nuclear localization control. (B) Transcriptional activity of SiNAC063, SiNAC070, and SiNAC118. The empty pGBKT7 vector was used as a negative control.