辣椒疫病（Phytophthora blight）由卵菌病原体 Phytophthora capsici 引起，是威胁辣椒生产的最严重病害之一，常导致大面积死棵和产量锐减。然而，辣椒中调控疫病抗性的关键转录因子及其作用机制尚不清晰。近日，安徽师范大学生命科学学院程伟教授团队在 Biology 发表了题为“Three NAC Transcription Factors, CaNAC61, CaNAC79, and CaNAC92, Negatively Regulate Pepper Resistance Against Phytophthora capsici”的研究论文。该研究通过转录组筛选和功能验证，首次鉴定出 CaNAC61、CaNAC79 和 CaNAC92 三个NAC转录因子在辣椒抗疫病中发挥负调控作用，为抗病育种提供了新的基因靶点。

研究团队基于前期辣椒-疫霉菌互作的RNA-seq数据，筛选出8个差异表达的NAC基因，并聚焦于 CaNAC61、CaNAC79 和 CaNAC92。RT-qPCR验证表明：CaNAC61 和 CaNAC79 表现为早期瞬时诱导，在接种后3小时（hpi）迅速达到峰值，随后下降；CaNAC92 则呈现持续晚期诱导，24 hpi后显著上调并维持高表达至72 hpi。这种差异化的时间动态提示这三个基因可能在防御反应的不同阶段发挥作用。系统发育分析显示，CaNAC61、CaNAC79和CaNAC92分别聚类到不同的NAC亚家族。多重序列比对证实三者均含有高度保守的N端NAC结构域（可细分为A-E五个亚结构域），C端则高度变异。亚细胞定位实验表明，三者的GFP融合蛋白均专一性地定位于细胞核，符合转录因子的典型特征。通过病毒诱导基因沉默（VIGS）技术分别沉默 CaNAC61、CaNAC79 或 CaNAC92 后，辣椒植株对 P. capsici 的抗性显著增强——离体叶片病斑直径明显减小，根部接种后病情指数也显著降低。相反，在辣椒叶片中瞬时过表达这三个基因中的任何一个，均导致病斑显著扩大，且防御标记基因 CaPR1（SA通路）、CaDEF1 和 CaLOX1（JA通路） 的转录水平被明显抑制。进一步在本氏烟中稳定过表达这三个基因，转基因植株同样表现出对疫霉菌的超感病性。这些结果一致证明：CaNAC61、CaNAC79 和 CaNAC92 是辣椒抗疫病的负调控因子。研究表明，这三个NAC转录因子可能通过抑制水杨酸（SA）和茉莉酸（JA）介导的防御信号通路来削弱植物免疫。值得注意的是，尽管三者均抑制CaPR1、CaDEF1和CaLOX1，但对CaPR2和CaPR10的影响存在差异（CaNAC61特异抑制CaPR2，CaNAC92特异抑制CaPR10），提示它们的功能并不完全冗余。

该研究补充了辣椒抗疫病转录调控网络中的“负调控模块”。负调控因子的存在对于防止免疫过度激活、维持正常生长发育具有重要生理意义。在育种应用中，精准下调或编辑这些负调控基因，有望成为提高辣椒疫病抗性的有效策略，为培育稳定、持久的抗病品种提供新思路。

Figure 1. Expression patterns of CaNAC61, CaNAC79, and CaNAC92 during P. capsici infection in pepper: (A) Transcriptional patterns of CaNAC61, CaNAC79, and CaNAC92 at 0, 3, 6, 12, 24, 48, and 72 h post-inoculation with P. capsici were determined by RNA-seq. (B) The expression patterns of CaNAC61, CaNAC79, and CaNAC92 at different time points post-inoculation were validated by RT-qPCR. Data are presented as the mean ± standard deviation (SD) from three independent biological replicates.

Figure 2. Phylogenetic relationships and conserved domain analysis of CaNAC61, CaNAC79, and CaNAC92: (A) A phylogenetic tree was constructed using NAC protein sequences from Capsicum annuum, Solanum lycopersicum, Nicotiana benthamiana, Glycine max, Arabidopsis thaliana, Oryza sativa, and Zea mays. CaNAC61, CaNAC79, and CaNAC92, which are highlighted in orange, were clustered into distinct phylogenetic clades with representative homologous NAC proteins from other plant species. Bootstrap values are represented by the size of the circles, and the scale bar indicates evolutionary distance. (B) Multiple sequence alignment was performed for CaNAC61, CaNAC79, and CaNAC92 with their homologous proteins. Sequence alignment revealed that all three proteins contain a conserved N-terminal NAC domain, a characteristic feature of the NAC transcription factor family that can be divided into five subdomains.

Figure 3. Subcellular localization of CaNAC61, CaNAC79, and CaNAC92. CaNAC61-eGFP, CaNAC79-eGFP, and CaNAC92-eGFP fusion proteins were transiently expressed in N. benthamiana leaves and co-expressed with the nuclear marker H2B-RFP. Confocal microscopy revealed that the GFP signals of CaNAC61, CaNAC79, and CaNAC92 strongly co-localized with the RFP signals, indicating that all three proteins are predominantly localized in the nucleus. eGFP was used as the empty vector control, showing fluorescence signals in both the nucleus and cytoplasm. GFP, green fluorescent protein. RFP, red fluorescent protein. Bar = 10 μm.

Figure 4. Silencing of CaNAC61, CaNAC79, and CaNAC92 enhances resistance to P. capsici in pepper: (A) Transcript levels of CaNAC61, CaNAC79, and CaNAC92 in TRV-mediated gene-silenced pepper plants were measured by RT-qPCR, with TRV: 0 serving as the negative control. Data represent the mean ± SD of three independent biological replicates. (B) Approximately 3–4 weeks post-agroinfiltration, TRV: PDS-treated plants exhibited typical photobleaching, confirming the effectiveness of the VIGS system. (C) Representative disease symptoms of detached VIGS leaves at 3 days post-inoculation (dpi) with P. capsici zoospores. (D) Lesion diameters after P. capsici inoculation at 3 dpi. (E) Representative disease symptoms of TRV-treated pepper plants subjected to root inoculation at 0 dpi and 6 dpi. (F) Disease indexes of these TRV-treated pepper plants after root inoculation with P. capsici zoospores. Data are presented as the mean ± SD from six independent biological replicates. Asterisks indicate significant differences based on Student’s t-test (* p < 0.05, ** p < 0.01).

Figure 5. Transient overexpression of CaNAC61, CaNAC79, and CaNAC92 enhances susceptibility of pepper leaves to P. capsici infection: (A) Transcript levels of CaNAC61, CaNAC79, and CaNAC92 in transiently overexpressing pepper leaves were measured by RT-qPCR. The empty vector (EV) served as the control. Data represent the mean ± SD of three independent biological replicates. (B) Lesion diameters in pepper leaves at 2 days post-inoculation (dpi) with P. capsici zoospores. Data are presented as the mean ± SD from six independent biological replicates. (C) Representative disease symptoms of pepper leaves following P. capsici inoculation at 2 dpi. (D) Expression of defense marker genes in pepper leaves transiently overexpressing CaNAC61, CaNAC79, or CaNAC92. Transcript levels of CaPR1, CaDEF1, and CaLOX1 were determined by RT-qPCR at 2 days post-agroinfiltration. Data were obtained from three independent biological replicates. The empty vector (EV) served as the control. All data are presented as the mean ± SD, and asterisks indicate significant differences based on Student’s t-test (* p < 0.05, ** p < 0.01).

Figure 6. Ectopic stable overexpression of CaNAC61, CaNAC79, and CaNAC92 enhances susceptibility to P. capsici in N. benthamiana: (A) RT-PCR analysis of CaNAC61, CaNAC79, and CaNAC92 transcript levels in transgenic N. benthamiana lines overexpressing each of these genes. NbActin was used as the internal reference, and wild-type (WT) plants served as the control.. (B) Representative disease symptoms of detached leaves from different transgenic N. benthamiana lines following P. capsici inoculation at 2 dpi. (C) Lesion diameters of these transgenic N. benthamiana leaves after P. capsici inoculation at 2 dpi. (D) Representative disease symptoms of these transgenic N. benthamiana plants subjected to root inoculation at 0 dpi and 7 dpi. (E) Disease indexes of different transgenic N. benthamiana lines after root inoculation with P. capsici zoospores. The data are presented as the mean ± SD from six independent biological replicates. Asterisks indicate significant differences based on Student’s t-test (** p < 0.01).

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