Abstract
Cyclic nucleotide-gated ion channels (CNGCs) have been reported to be involved in multiple plant physiological processes. Their involvement in plant immunity has been studied in several herbal plant species. It remains unclear whether CNGCs in woody plants play a similar role in plant immunity. In the present study, we identified an apple CNGC (designated as MdCNGC2), which is the homolog of Arabidopsis CNGC2. Analysis of tissue distribution revealed that MdCNGC2 was expressed in all tested tissues. Abundant transcripts of MdCNGC2 were observed in leaves and shoot bark. Low expression was observed in fruits and roots. MdCNGC2 expression was induced in apple callus and shoot bark by Botryosphaeria dothidea. The induction of MdCNGC2 was significantly higher in susceptible cultivars “Fuji,” “Ralls Janet,” and “Gala” compared to the resistant cultivar “Jiguan,” suggesting that MdCNGC2 may be a negative regulator of resistance to B. dothidea. MdCNGC2 mutagenesis mediated by gene editing based on the CRISPR/Cas9 system led to constitutive accumulation of SA in apple callus. A culture filtrate of B. dothidea (BCF) induced the expression of several defense-related genes including MdPR1, MdPR2, MdPR4, MdPR5, MdPR8, and MdPR10a. Moreover, the induction of these genes was significantly higher in mdcngc2 mutant (MUT) callus than in wild type (WT) callus. Further analysis showed that the spread of B. dothidea was significantly lower on MUT callus than on WT callus. Knockdown of the MdCNGC2 gene reduced lesions caused by B. dothidea in apple fruits. These results collectively indicate that MdCNGC2 is a negative regulator of resistance to B. dothidea in apple callus.
Highlights
During infection by pathogenic microorganisms, a change in the intracellular ion flux is one of the earliest responses of plant cells (Moeder et al, 2011)
A phylogenetic tree was constructed using Cyclic nucleotide-gated ion channels (CNGCs) proteins (Supplementary Table S2) from Arabidopsis, rice, tomato, and apple (Figure 1B). These proteins were divided into five groups, and MdCNGC2 clustered with AtCNGC2, AtCNGC4, OsCNGC14, OsCNGC15, and OsCNGC16; most of these proteins are reported to be associated with plant disease resistance (Ali et al, 2007; Chin et al, 2013; Nawaz et al, 2014)
CRISPR/Cas9-mediated mutagenesis of MdCNGC2 increased apple callus resistance to B. dothidea, and MdCNGC2 silencing in fruits improved the resistance to B. dothidea
Summary
During infection by pathogenic microorganisms, a change in the intracellular ion flux is one of the earliest responses of plant cells (Moeder et al, 2011). Pathogen infection causes fluctuations in Ca2+ flux and leads to immune responses warding off invaded pathogens In this process, the calcium channels play crucial roles. At least four Arabidopsis CNGCs have been reported to be crucial for defense responses to pathogen infection, i.e., AtCNGC2 (Ali et al, 2007), AtCNGC4 (Chin et al, 2013), AtCNGC11, and AtCNGC12 (Yoshioka et al, 2001, 2006). A rice CNGC was reported to play a crucial role in blast resistance (Wang et al, 2019). All these studies indicate that CNGCs are important for plant disease resistance
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