CN-121975842-A - Method for enhancing plant disease resistance based on rice disease resistance PTR protein and application

Abstract

The invention provides a method for enhancing plant disease resistance based on rice disease resistance PTR protein and application thereof. The rice disease-resistant PTR protein is an endoplasmic reticulum-localized pentameric Ca 2+ channel, and the C terminal end of the protein comprises a self-inhibition domain, wherein the domain contains a CaaX motif (CCIS), and the C902 cysteine is modified by farnesylation to maintain the channel inactivated state. The rice blast effector protein AVR-Pita specifically recognizes and cleaves the site (PEPPA ∈ KPEP) between alanine 874 (a 874) and lysine 875 (K875) of PTR, removes the self-inhibiting module, activates its Ca 2+ channel activity, and releases Ca 2+ of the endoplasmic reticulum to the cytoplasm, further triggering an immune response. The method can be widely applied to disease-resistant breeding of rice and other crops, and has good application prospect and industrial value.

Inventors

• CHAI JIJIE
• GE DONGDONG
• HE YANG
• HAN ZHIFU

Assignees

• 西湖大学

Dates

Publication Date

20260505

Application Date

20260206

Claims (10)

1. 1. A method for enhancing plant disease resistance based on rice disease-resistant PTR protein is characterized in that the self-inhibition state of the PTR protein is relieved by knocking out, replacing or genetically editing the C-terminal sequence of the rice disease-resistant PTR protein, and the PTR-mediated endoplasmic reticulum calcium ion channel function is activated, so that the resistance of plants to pathogenic bacteria is enhanced.
2. 2. The method according to claim 1, wherein the rice disease-resistant PTR protein is a pentameric Ca 2+ channel protein, the important amino acids of the channel activity comprise 124 th site aspartic acid and/or 198 th site aspartic acid, and the amino acid sequence is shown in SEQ ID NO. 1.
3. 3. The method of claim 1, wherein the C-terminal sequence comprises a self-inhibitory domain and a CaaX lipid modification motif, wherein the CaaX lipid modification motif is CCIS.
4. 4. A method according to claim 3, wherein the amino acid sequence of the self-inhibiting domain is as shown in SEQ ID No.11 and the cysteine at position 902 of the C-terminal end is subjected to a farnesylation modification.
5. 5. The method of claim 1, wherein the method of relieving the self-inhibited state of PTR protein is selected from one of the following: (1) Deletion or mutation rice disease-resistant PTR protein or self-inhibition structural domain of the C terminal of the disease-sensitive PTR protein, wherein the amino acid sequence of the disease-sensitive PTR protein is shown as SEQ ID NO.10, and the amino acid sequence of the self-inhibition structural domain is shown as SEQ ID NO. 11; (2) Disruption or replacement of rice disease-resistant PTR proteins and CaaX lipid modifications at the C-terminus of the disease-resistant PTR proteins; (3) Mutation, substitution and substitution of lysine for arginine at 879 of rice-induced PTR protein; (4) And cutting the disease-resistant PTR of the rice.
6. 6. The method of claim 5, wherein the cleaving rice disease-resistant PTR protein is cleavable by a pathogen effector protein AVR-Pita, the cleavage site being located between alanine 874 and lysine 875 of the PTR protein.
7. 7. The activatable plant immunity channel protein for enhancing plant disease resistance is characterized in that the amino acid sequence of the rice disease resistance PTR protein is shown as SEQ ID NO.1, SEQ ID NO.12 and SEQ ID NO. 13.
8. 8. A method for enhancing disease resistance in a plant comprising introducing into the plant any one of the proteins of claim 7.
9. 9. The method for enhancing plant disease resistance based on rice disease-resistant PTR protein as set forth in claim 1 to 6, or the activatable plant immunity channel protein for enhancing plant disease resistance as set forth in claim 7, or the application of the method for enhancing plant disease resistance as set forth in claim 8 in enhancing plant immunity and disease resistance.
10. 10. The use according to claim 9, wherein the plant is rice, wheat, maize or barley.

Description

Method for enhancing plant disease resistance based on rice disease resistance PTR protein and application Technical Field The invention belongs to the technical fields of plant molecular immunology and crop molecular breeding, and particularly relates to a method for enhancing plant disease resistance based on rice disease resistance PTR protein and application thereof. Background Rice (Oryza sativa L.) is one of the most important food crops in China and even worldwide, and the yield and quality of the rice are directly related to the food safety. However, in the course of rice growth and development, rice blast (Magnaporthe oryzae causes) occurs widely, and when serious, significant yield reduction can be caused, and significant economic loss is brought. Therefore, the excavation and utilization of the rice blast resistance gene to cultivate excellent varieties with stable and durable resistance is the most economical and effective technical approach for preventing and treating rice blast. At present, the cloned and applied rice blast resistance genes are mainly NLR (nucleic acid-binding Leucine-RICH REPEAT) disease resistance genes, such as Pi54, pik, pita and the like. Such genes activate downstream immune responses, usually by specifically recognizing pathogenic effector (e.g., avrPi, avrPik, AVR-Pita), conforming to the classical "gene-to-gene" resistance pattern. However, NLR disease-resistant mechanisms have obvious defects of strong resistance specificity and poor broad spectrum, are often effective on specific pathogenic species, are easy to break through, can be rapidly escaped and identified by pathogenic bacteria through effector sequence variation (such as single nucleotide polymorphism of AVR-Pita), have short resistance duration, and often have the phenomenon that the resistance is maintained for only 3-5 years in production. Therefore, analyzing the non-NLR disease-resistant mechanism and developing disease-resistant gene resources with broad spectrum and durability has become the leading edge of international research in the field of crop disease-resistant breeding. Up to now, researchers at home and abroad have identified more than 500 NLR genes in rice, of which the number of specific rice blast resistant NLR genes of the race that were successfully cloned and functionally verified has been more than 50. Pita/AVR-Pita is one of the earliest reported pair of rice NLR/effector combinations. It has been shown that AVR-Pita encodes a metalloprotease containing a zinc ion binding site, which, upon secretion into rice cells, binds to a specific domain of Pita protein in the C-terminal region, thereby inducing an immune response (Jia et al, 2000; orbach et al, 2000). However, recent genetic studies have shown that the Pita protein itself does not directly mediate the recognition process of AVR-Pita. Several lines of evidence suggest that AVR-Pita-induced immune responses are independent of classical Pita signaling pathways, but rather rely on another PTR (Pita-associated protein) -mediated signaling system (Xiao et al, 2024). The PTR gene is located on chromosome 12 of rice and forms a tightly linked gene cluster with the Pita resistance gene. It has been reported that PTR encodes an atypical disease-resistant protein whose protein structure comprises an ARM repeat domain and a transmembrane region, and differs significantly in structure from conventional NLR proteins (Zhao et al, 2018). Although genetic evidence has clarified that PTR plays a key role in AVR-Pita mediated disease resistance, the prior art still has the defects that whether the molecular action mechanism of PTR protein is not clear, whether the PTR protein directly recognizes a pathogenic effector, how to activate immune signal lack system description, whether PTR has ion channels or other biochemical functions and action sites in cells, whether the prior study does not give clear conclusion, whether PTR protein has a self-inhibition regulation mechanism, the structural functional elements and the regulation mode of the PTR protein are not resolved, and no systematic study on the key structural domain, the regulation mechanism or the application of the PTR protein in crop disease resistance engineering exists at present. Therefore, on the basis of the existing research, a new PTR-mediated non-NLR disease-resistant mechanism is deeply analyzed, the key functional structure and regulation mode of the mechanism are clarified, and the application potential of the mechanism in broad-spectrum and durable disease-resistant breeding of crops is explored. Disclosure of Invention Aiming at the defects existing in the prior art, the invention provides a method for enhancing plant disease resistance based on rice disease resistance PTR protein and application thereof. In order to achieve the above purpose, the invention adopts the following technical scheme: In a first aspect, the present invention provides a method for enhancing plant di