Functional Analysis of Wheat NAC Transcription Factor, TaNAC069, in Regulating Resistance of Wheat to Leaf Rust Fungus

Abstract

NAC transcription factors are one of the largest transcription factor families having functions in a variety of stress responses. Few NACs have been reported for interactions between wheat and the wheat rust fungus Puccinia triticina (Pt). In this study, based on analysis of RNA-seq data from wheat line TcLr19 inoculated by Pt, the NAC transcription factor TaNAC069 was cloned from wheat, and its transcriptional activity and homologous dimer formation were verified. Quantitative real-time PCR analysis showed that the expression of TaNAC069 was induced by Pt and associated signaling molecules. To further characterize the function of the TaNAC069 gene in wheat resistance to Pt, virus-induced gene silencing (VIGS) was utilized, and it revealed that Pt resistance in TaNAC069-silenced plants was significantly reduced. Potential interaction targets of TaNAC069 from wheat and Pt were screened and identified by yeast two-hybrid technology. Eukaryotic elongation factor eEF1A, CBSX3 protein, and cold acclimation protein WCOR410c were screened by yeast one-hybrid technology. The results indicate that the TaNAC069 gene plays a positive regulatory role in wheat resistance to Pt, laying a good foundation to analyze the molecular mechanisms of TaNAC069 and its functional role in wheat resistance to Pt.

Keywords: NAC transcription factor; Puccinia triticina; RNA-seq; VIGS; clone.

FIGURE 1

Analysis of TaNAC069 gene. (A) Structural domains of TaNAC069. (B) Multiple alignments of TaNAC069 and homologous genes. (C) Phylogenetic analysis of NACs. NAC subdomains (A–E) are conserved and underlined. Identical and similar residues are shaded in different colors, respectively. The unrooted phylogenetic tree of NAC domains was depicted using the CLUSTAL X program and was constructed using the neighbor-joining method. ANAC001, Arabidopsis thaliana NAC001 (AT1G01010); NAC1, A. thaliana NAC1 (AF198054); NAC2, A. thaliana NAC2 highly expressed in root meristem (AF201456); AtNAC3, A. thaliana NAC3 (AB049070); ANAC011, A. thaliana NAC011 (AT1G32510); ANAC063, A. thaliana NAC063 (AT3G55210); ATAF1, Arabidopsis transcription activation factor 1 (X74755); TIP, A. thaliana TIP interacts with turnip crinkle virus capsid protein (AF281062); ONAC001, Oryza sativa NAC001 (AK060509); ONAC003, O. sativa NAC003 (AK061716); OsNAC3, O. sativa NAC3 (AB028182); OsNAC7, O. sativa NAC7 (AB028186); OsNAC8, O. sativa NAC8 (AB028187); ONAC022, O. sativa NAC022 (AK107090); TERN, tobacco elicitor-responsive (AB021178); NAM, Petunia hybrida no apical meristem (X92204); NAP, NAC-like, activated by AP3/PI (AJ222713); SENU5, Lycopersicon esculentum SENU5 (Z75524).

FIGURE 2

Analysis of TaNAC069 gene expression. (A) Expression profiles of the TaNAC069 gene in wheat infected with Pt. (B) Expression profile of the TaNAC069 gene in different organs. The y-axis indicates the amounts of gene transcripts normalized to the GAPDH gene and expressed relative to that of mock-inoculated control plants treated with 0.1% (v/v) ethanol solution. The x-axis indicates different sampling. The reference gene is GAPDH. The assays for each treatment consisted of three biological replicates. Data are means ± standard errors of three independent experiments. Differences between time-course points were assessed using SSPS. ^∗p < 0.05, n = 3.

FIGURE 3

Expression profiles of the TaNAC069 gene in response to different chemical inducers. The y-axis indicates the amounts of gene transcripts normalized to the GAPDH gene and expressed relative to mock-inoculated control plants treated with 0.1% (v/v) ethanol solution. The x-axis indicates different sampling timepoints. The reference gene is GAPDH. The assays for each treatment consisted of three biological replicates. Data are means ± standard errors of three independent experiments. Differences between time-course sampling points were assessed using SSPS. ^∗p < 0.05, n = 3.

FIGURE 4

Functional analysis of the TaNAC069 gene in response to leaf rust infection using virus-induced gene silencing. (A) Disease symptoms of TaNAC069 gene-knockdown wheat leaves 14 days after inoculation with Pt pathotype PHNT. (B) Relative expression of wheat TaNAC069 in gene-knockdown wheat leaves after inoculation with PHNT. Mock, TcLr19 wheat leaves without barley stripe mosaic virus (BSMV) and PHNT inoculation; BSMV:00, TcLr19 after BSMV and PHNT inoculation; Tc+, Thatcher after PHNT inoculation. The reference gene is GAPDH. The assays for each treatment and phenotype combination consisted of at least three biological replicates (BSMV:00 was used as the control). Data are means ± standard errors of three independent experiments. Differences between time-course sampling points were assessed using SSPS. ^∗p < 0.05, n = 3.

FIGURE 5

Relative transcript levels of TaPR1, TaPR2, TaTLP1, TaSOD, TaCAT, and TaNOX in TaNAC069-knockdown plants inoculated with PHNT. The y-axis indicates the amounts of gene transcripts normalized to the GAPDH gene and expressed relative to that of mock-inoculated control plants treated with 0.1% (v/v) ethanol solution. The x-axis indicates different sampling timepoints. The reference gene is GAPDH. The assays for each gene consisted of at least three biological replicates. Data are means ± standard errors of three independent experiments. Differences between time-course sampling points were assessed using SSPS. ^∗p < 0.05, n = 3.

FIGURE 6

Determination of leaf H₂O₂ concentration by 3,3-diaminobenzidine (DAB) staining. (A) H₂O₂ accumulation in TaNAC069-knockdown wheat plants was detected by staining with DAB and viewed under differential interference contrast optics. Scale bar, 50 μm. (B) Superoxide dismutase (SOD) and catalase (CAT) activity in TaNAC069-knockdown wheat plants. The y-axis indicates the amount of SOD and CAT activities (each treatment and phenotype combination consisted of at least three biological replicates). The x-axis indicates different sampling timepoints. Data are means ± standard errors of three independent experiments. Differences between time-course sampling points were assessed using SSPS. ^∗p < 0.05, n = 3 (BSMV:00 used as the control).

FIGURE 7

Identified activity of TaNAC069 and pTaNAC069. (A) Transcription activation activity of the TaNAC069. (B) Homocomplex formation of the TaNAC069 in yeast. (C) GUS histochemical staining directed by the pTaNAC069 sequences in tobacco. pBI121, GUS histochemical staining results of tobacco leaves transformed by vector pBI121; pBI121-pTaNAC069, GUS histochemical staining results of tobacco leaves transformed by pBI121-pTaNAC069; CK, GUS histochemical staining results of tobacco. Scale bars are 50 μM. (D) GFP expressed by the pTaNAC069 sequences in tobacco. M, Marker 2000; 1–2, restrictive digestion of the pCamA-pTaNAC069-GFP plasmid; pCamA-GFP, results of tobacco leaves transformed by vector pCamA; pCamA-pTaNAC069-GFP, results of tobacco leaves transformed by pCamA-pTaNAC069-GFP. Scale bars are 50 μM.

FIGURE 8

Interaction between TaNAC069 and TaCP5. (A) Yeast two-hybrid analysis of the interaction between TaNAC069 and TaCP5 in yeast. Yeasts expressing the indicated combinations of bait and prey proteins were spotted onto synthetic dropout medium lacking leucine and tryptophan (SD-WL) and SD medium lacking leucine, tryptophan, histidine, and adenine supplemented with X-α-Gal and Aureobasidin A (SD-HAWL + X-α-Gal). Only yeast strains co-expressing TaNAC069 and TaCP5 grew on SD-HAWL plates and developed blue coloration, indicative of an interaction between both proteins. TaPR1 and TaTLP1 are the positive controls; TaNAC069-N and pGADT7, TaCP5 and pGBKT7, and pGADT7 and pGBKT7 were used as negative controls. (B) Gene expression profiles of TaNAC069 and TaCP5 in wheat infected with Pt at different timepoints post-inoculation. The y-axis indicates the amounts of gene transcripts normalized to the GAPDH gene and expressed relative to plants at 0 hpi. The x-axis indicates different sampling timepoints. The reference gene is GAPDH. The assays for each treatment included three biological replicates. Data are means ± standard errors of three independent experiments. Differences between time-course sampling points were assessed using SSPS. *p < 0.05, n = 3.