Summary
Melatonin is known to play a pivotal role in plant immunity, but the regulation of melatonin production during pathogen infection is largely unknown.
The regulatory role of receptor-like cytoplasmic kinase MdPBL34 in regulating melatonin synthetic enzyme MdSNAT5 and its interaction with partner MdGRF3 was explored by biochemical analyses. The stable transgenic apple (Malus domestica Borkh.) lines with overexpressed and RNA-interfered MdSNAT5, as well as transiently transformed apple leaves with overexpressed MdGRF3 or RNA-interfered MdPBL34, were generated. MdSNAT5 phosphorylation by MdPBL34 and its stabilization by MdGRF3 by reducing ubiquitination was examined.
We revealed Alternaria alternata f. sp. Mali infection promoted melatonin production, which further enhanced apoplastic reactive oxygen species (ROS) burst. Overexpression of MdSNAT5 conferred transgenic apple plants enhanced Alternaria blotch resistance. The pathogen attack activates MdPBL34 kinase, which phosphorylates MdSNAT5 specifically at Ser148. Phosphorylation significantly enhances MdSNAT5 enzyme activity and facilitates its interaction with MdGRF3. MdGRF3 stabilizes MdSNAT5 by decreasing its ubiquitin-mediated degradation for increased melatonin levels. The role of the MdPBL34-MdSNAT5-MdGRF3 pathway was confirmed by employing a specific phospho-Ser148 antibody across several tolerant and susceptible apple cultivars.
Our findings offer valuable insights and practical strategies for breeding disease-resistant apple cultivars and developing melatonin-based sustainable disease management approaches.
概括
褪黑素在苹果斑点落叶病免疫中起关键作用，但调控机制尚不清楚。该研究首次证明了褪黑素能够提高RBOH酶活，触发质外体活性氧(ROS)爆发，抵御斑点落叶病菌。过表达褪黑素合成限速酶基因MdSNAT5，显著转基因苹果植株的褪黑素水平和抗病性。被斑点落叶病菌激活的胞质类受体激酶MdPBL34，特异性磷酸化MdSNAT5的Ser148位点，显著提升它的酶活，更促进了它与MdGRF3的互作，进而减少MdSNAT5的泛素化降解，提高褪黑素水平和斑点落叶病抗性。最后，利用特异性的pSer148抗体在多个苹果抗/感品种中证实了MdPBL34-MdSNAT5-MdGRF3调控通路的存在。该研究为培育强化褪黑素的抗病苹果品种，以及开发基于褪黑素的绿色病害防控策略提供了理论依据。
