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Literature Sharing | CsNAC17 enhances resistance to Colletotrichum gloeosporioides by interacting with CsbHLH62 in Camellia sinensis
Release time:
2025-02-17
The pathogen Colletotrichum gloeosporioides causes anthracnose, a serious threat to tea trees around the world, particularly in warm and humid regions. RNA-Seq data have previously indicated NAC transcription factors are involved in anthracnose resistance, but underlying mechanisms remain unclear. The BiFC, Split-LUC, and Co-IP assays validated the interaction between CsbHLH62 and CsNAC17 identified through yeast two-hybrid (Y2H) screening. CsNAC17 or CsbHLH62 overexpression enhanced anthracnose resistance, as well as enhanced levels of H2O2, hypersensitivity, and cell death in Nicotiana benthamiana. The NBS-LRR gene CsRPM1 is regulated by CsNAC17 by binding directly to its promoter (i.e. CACG, CATGTG), while CsbHLH62 facilitates CsNAC17’s binding and increases transcriptional activity of CsRPM1. Additionally, transient silencing of CsNAC17 and CsbHLH62 in tea plant leaves using the virus-induced gene silencing (VIGS) system resulted in decreased resistance to anthracnose. Conversely, transient overexpression of CsNAC17 and CsbHLH62 in tea leaves significantly enhanced the resistance against anthracnose. Based on these results, it appears that CsbHLH62 facilitates the activity of CsNAC17 on CsRPM1, contributing to increased anthracnose resistance.
Colletotrichum sp. causes anthracnose, a damaging disease that threatens Camellia sinensis (L.) O. Kuntze cultivation. While the pathogen primarily infects young tissues, symptoms are more pronounced on mature leaves due to extended incubation periods. The deleterious effects of anthracnose on yield and quality underscores the need for effective prevention and control measures. Presently, on account of commonly used chemical agents that raise concerns about potential threats to food security, the study on related disease-resistance genes offers a foundation for enhancing disease resistance in tea plants via breeding or genetic modification.
The pathogenicity test demonstrated that the characteristic anthracnose brown spot manifested on the ‘LJ43’ leaves during pathogen infection at 3 and 4 days post-inoculation (dpi), but with no obvious disease symptoms on the ‘ZC108’ leaves (Fig. 1a). Aniline blue staining techniques was applied to examine the infection structures of the fungi C. gloeosporioides in tea plant leaf tissues, which revealed that the development of C. gloeosporioides on ‘LJ43’ leaves was faster than that on ‘ZC108’ (Fig. 1b). Trypan blue staining revealed apparent significant HR formation on ‘ZC108’ leaves during C. gloeosporioides infection, while no such response was observed on ‘LJ43’ leaves. Furthermore, ‘ZC108’ leaves showed epidermal cell necrosis, whereas only mesophyll cell necrosis was noted in ‘LJ43’ leaves, but no evidence of epidermal cell necrosis (Fig. 1c). The production and accumulation of H2O2 was observed by DAB staining. The results demonstrated a significantly greater accumulation of H2O2 on ‘ZC108’ leaves than that on ‘LJ43’ leaves (Fig. 1d). These findings confirmed that ‘ZC108’ is more resistant to C. gloeosporioides compared to ‘LJ43’. Combined with our previous transcriptome data in ZC108 after C. gloeosporioides infection , we proposed CsNAC17 as an important resistance factor that was further proved by real-time quantitative polymerase chain reaction (RT-qPCR).
Figure 1. Histochemical observation of ‘Longjing 43’ (LJ43) and ‘Zhongcha 108’ (ZC108) inoculated with C. gloeosporioides.
Tobacco lines overexpressing CsNAC17 (OE-CsNAC17) were constructed to investigate its role in fungal resistance. Following independent transformation (Fig. 3b; Supplemental Fig. S2a), homozygous transgenic plants were identified and three lines (i.e. L2, L3) with elevated CsNAC17 expression levels were selected. Inoculation tests of OE-CsNAC17 tobacco T3 generation lines and wild-type (WT) by C. gloeosporioides revealed small water-soaking spots in WT leaves at 4 dpi, while transgenic plant leaves had no obvious changes. Notably, a gradual increase in lesion area occurred in WT leaves between 7 and 10 dpi, whereas transgenic leaves exhibited no such alterations (Fig. 3a). Trypan blue staining illustrated limited or just initiated germination of C. gloeosporioides spores in both WT and transgenic lines at 1 dpi. The hyphae of the pathogen in the WT leaves were notably longer, while transgenic lines displayed shorter hyphae at 4 dpi (Fig. 3c). The hyphae congregated in the WT leaves, whereas the hyphae growth in the transgenic lines was hindered, with the haustoria being dried and shriveled from 7 dpi (Fig. 3c). WT leaves harbored numerous conidia, with hyphae covering the entire leaves, while transgenic lines exhibited no significant changes in pathogens at 10 dpi (Fig. 3c). DAB staining revealed a higher H2O2 accumulation in the transgenic leaves compared to the WT at 4 dpi (Fig. 3d), which was consistent with the observed change in H2O2 content determined by a kit (Fig. 3e), suggesting a stronger HR response occurred in the OE-CsNAC17 lines compared to the WT.
Figure 3. Overexpressing CsNAC17 in tobacco enhances resistance to anthracnose
CsNAC17 resistance mechanism was investigated using the Y2H assay. BD-CsNAC17 was used as bait for screening the cDNA library and eight clones were found. Among them, CsbHLH62, a transcription factor belonging to the bHLH family, known for its role in plant disease resistance, was selected for further analysis. As with the positive control, yeast colonies harboring BD-CsNAC17 and AD-CsbHLH62 grew on SD/−Ade-His/Leu-Trp medium and displayed a blue coloration (Fig. 4a). The results confirmed the specific interaction between CsNAC17 and CsbHLH62 in yeast cells.
Figure 4. Interaction analysis of CsNAC17 and CsbHLH62
The CsbHLH62 CDS from ‘ZC108’ was cloned and sequenced. Sequence analysis revealed only one amino acid variation, with a remarkable 99.8% similarity between ‘ZC108’ and ‘LJ43’ (Fig. S1e). CsbHLH62 possessed an ORF of 1521 bp and was localized in the nucleus (Fig. 5a). RT-qPCR analysis revealed significant upregulation of CsbHLH62 transcription both in ‘LJ43’ and ‘ZC108’ leaves at 72 hpi with C. gloeosporioides (Fig. 5b). Subsequently, tobacco lines overexpressing CsbHLH62 (OE-CsbHLH62) were established to select high-expression lines L2 and L4 for further investigation (Fig. 5c; Supplemental Fig. S2b). The morphological analysis demonstrated that WT exhibited lesions 4 and 7 dpi, while the transgenic lines displayed no obvious symptoms (Fig. 5d). Trypan blue staining at 1 dpi revealed no significant disparity in leaf morphology between OE-CsbHLH62 and the control, whereas a notable distinction was observed at 7 dpi. OE-CsbHLH62 had limited hyphae compared to extensive coverage on WT leaves
(Fig. 5e). After 4 dpi, we observed an obvious HR in OE-CsbHLH62 lines, which was absent in the WT (Fig. 5f). Moreover, DAB staining indicated higher H2O2 accumulation in infected OE-CsbHLH62 leaves compared to WT at 1 and 7 dpi (Fig. 5g). The H2O2 content indicated that H2O2 accumulation in OE-CsbHLH62 lines was higher than that in WT after infection with C. gloeosporioides (Fig. 5h), suggesting that overexpressing CsbHLH62 in tobacco enhanced defense response and resistance to anthracnose.
Figure 5. CsbHLH62 transgenic tobacco exhibits enhanced resistance to anthracnose.
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