Function Study Of E3 Ubiquitin Ligase CTI1 And RHE In Meloidogyne Incognita Resistance And Cadmium Stress Resistance In Tomato Respectively

In recent 30 years,horticultural facilities have achieved rapid development and play an important role in ensuring vegetable supply and improving national health in China.However,the continuous cropping barriers,application excessive chemicals,diseases,insect pests and heavy metal pollution of soil become increasingly serious,which directly threaten the sustainable development of horticulture facility and the safety of agricultural products in China.Therefore,it is of great practical and scientific significance to study and seek effective methods to overcome the obstacles of continuous cropping,pollution along with the development of horticulture facility soil in China.E3 ubiquitin ligase is one of the key enzymes that constitute the ubiquitin-proteasome(UPS)degradation system and is widely involved in diverse plant growth and development processes and adversity response.The mechanism of E3 ubiquitin ligase CTI1(CSN-Interacting-Protein 1)mediated tomato resistance to RKN(Root-knot nematode)was studied.The important function of E3 ubiquitin ligase RHE(Root-High-Expression protein)in tomato towards cadmium stress resistance was preliminarily revealed.The main research results are as follows:1.We have found that RING family E3 ubiquitin ligase CTI1(Solyc01g105620)mediated tomato resistance to RKN.When RKN infect tomato roots,E3 ubiquitin ligase CTI1 gene expression was induced rapidly in tomato.CTI1 gene knockout mutants cti1 obtained by CRISPR/Cas9 showed sensitivity to RKN compared with wild-type(WT),while overexpressed CTI1 strains showed enhanced resistance to RKN infection.After RKN treatment,the content of jasmonic acid(JA)and jasmonate-isoleucine(JA-Ile)in cti1 plants was lower than that in WT plants,while the content of JA,JA-Ile and the transcript levels of JA-related genes in overexpressed plants were higher than that in WT plants,indicating that CTI1 regulates RKN resistance in tomato through the JA pathway.In addition,we alao found that CTI1 and COP9 signaling complex subunits 4(CSN4)and CSN5 A interact in vivo through Bi FC and Co-IP,respectively.These results suggest that E3 ubiquitin ligase CTI1 interacts with the COP9 complex by activating the JA signaling pathway and participates in basal resistance to RKN infection in tomato.2.We have studied the mechanism of COP9 signaling complex subunits CSN4 and CSN5 mediating RKN resistance.After infection with RKN,transcription levels of CSN4 and CSN5 along with their protein levels induced rapidly in tomato roots.Silencing of CSN4 and CSN5 by virus-induced gene silencing(VIGS)compromised the resistance towards RKN,subsequently increasing their number of egg masses.It was further observe that the number of root nodes in the root system along with lipid peroxidation of the cell membrane significantly increased compared to WT after RKN infection in gene silenced tomato plants.More importantly,the accumulation of JA content and the expression of JA biosynthesis and signaling related genes were also significantly inhibited in CSN4 and CSN5 silenced plants.In addition,protein interaction experiments showed that CSN4 and CSN5 B could interact with JA signaling inhibitor JAZ2;however,RKN induced JAZ2 expression was also inhibited in CSN4 or CSN5 silenced plants.In summary,our results suggest that tomato CSN4 and CSN5 play a key role in RKN basal defense that relies on JA signaling pathways..3.We have identified RING family E3 ubiquitin ligase RHE(Solyc09g089890)mediated cadmium tolerance in tomato plants.By transcriptional analysis,we found that E3 ubiquitin ligase gene RHE had a high expression response to cadmium stress.Therefore,we then constructed the tomato RHE gene over-expressing and mutant lines to verify its role.Compared with WT,RHE over-expressing plants have shown significant tolerance to cadmium stress as its reduces Cd accumulation,and by significantly improving the 20 S proteasome activity.However,rhe mutants exhibited the opposite phenotypes.It was noteworthy to mentioned that over-expressing of RHE gene in tomato plants significantly lowers the Cd mediated accumulation of reactive oxygen species(ROS)and improves the overall antioxidant potential in tomato plants.In addition,compared with WT,the amount of cadmium accumulation significantly increased in rhe mutant plants.This might be interrelated with the differential expression of CAX3(Calcium/proton exchanger 3),IRT1(Iron from the protein 1),HMA(Heavy-metal-translocating ATPase)in RHE over-expressing and rhe mutants plants.In conclusion,RHE over-expression in tomato plants may enhance cadmium tolerance by Ub/26 S proteasome thus scavenging the cadmium-damaged proteins and by increasing the antioxidant enzyme activity thus reduces oxidative stress in tomato plants.The low cadmium accumulation in RHE over-expressing plants may attributed due to the high expression of CAX3 and the low expression of IRT1 and HMA.Based on these findings,the conclusion is as follows:1.The RING family E3 ubiquitin ligase CTI1 positively regulates the resistance to RKN,and regulates the RKN induced JA signalosome through interaction with the subunits CSN4 and CSN5 A of COP9 signalosome complex.COP9 signalosome complex subunits CSN4 and CSN5 A are necessary for JA synthesis and signaling pathway after RKN infection,and JAZ2 interacts with COP9 signal complex subunit CSN4/CSN5 B to participate in CSN-mediated RKN resistance.2.The RING family E3 ubiquitin ligase RHE positively regulates cadmium stress resistance by increasing 26 S proteasome activity and antioxidant defense system,and is affected by cadmium transport-related genes expression.