Study On The Communication Between Dodder And Host Plant And Overexpression Of CuRe1 Confers Resistance To Dodder In Alfalfa

In nature,dodders(Cuscuta spp.)are holoparasites that consist of about 201 species,which are widely distributed all over the world.After the establishment of the parasitic relationship,the exchange of water,nutrient,DNA,mRNA,mi RNA,and protein in dodder–host plant,which is thus used as an ideal model for the study of communication between plants in parasitic systems.However,dodders are parasitic weeds with highly destructive and difficult control,which seriously endangers the growth and production of forage and crops.In the past ten years,with the help of omics sequencing technology,complex communication has rapidly become an important academic frontier in parasitic systems,but breeding forages with resistance to parasites is still blank in the world.Due to the alfalfa lacked genomic information when the experiment was started,we used the dodder and soybean(Glycine max)system to study the characteristic of lncRNAs(long noncoding RNAs)and found that lncRNAs were translocated between soybean and dodder.Subsequently,the dodder resistance gene CuRe1(Cuscuta receptor 1)was overexpressed into alfalfa(Medicago sativa)and conferred alfalfa resistance against dodder.The resistance phenotype,haustorial characteristics,and transcriptional responses of CuRe1-overexpressed alfalfa were analyzed.Finally,a field trial was carried out on CuRe1-overexpressed alfalfa for three consecutive years to evaluate the phenotypic and physiological changes of alfalfa after dodder parasitism,and stably genetic dodder-resistant alfalfa was obtained.The transgenic materials created in this study provided a basis for breeding alfalfa resistant to dodder.The main research results are as bellow:1.Bidirectional lncRNA transfer between dodder parasites and their host plant.In this study,the dodder and soybean were used to perform lncRNA sequencing,genomewide analysis,and RT-PCR verification.The results showed that lncRNAs could be translocated within the dodder–soybean parasitic system,among which 365 dodder and14 soybean lncRNAs were found in soybean and dodder stems,respectively.The abundance may be the major driving force for the inter-plant mobility of macromolecules.Furthermore,we found that many lncRNA-mRNA interaction networks could be co-transferred between dodder and host soybean.2.Function analysis of tomato CuRe1 against dodder parasitism in alfalfa host.To study the common and special characteristics of host plants’ resistance to dodder,CuRe1 was overexpressed in three different species,respectively.Compared to the dodders parasitized on the control alfalfa plants(Wild-type and empty vector),we found that the three CuRe1-overexpressed alfalfa lines showed resistance against dodder parasitism,accompanying dark and shrunk haustorium and unnormal connection of vascular bundle.After dodder parasitism,CuRe1-overexpressed alfalfa plants exhibited a significant increase in the biomass than those of the control plants,while the dodders parasitized CuRe1-overexpressed alfalfa plants showed a lower biomass than the dodders parasitized on the control alfalfa plants.When the CuRe1 gene was overexpressed in tobacco(Nicotiana benthamiana)and Arabidopsis(Arabidopsis thaliana),the growth of dodder on transgenic plants was dramatically inhibited,exhibiting strong resistance to dodders.Combined with transcriptomic data from three species of alfalfa,tobacco,and Arabidopsis,we found that CuRe1 could recognize dodder as a pathogen,induce PTI(pattern-triggered immunity)-related immunity of host plants,and trigger the expression of a series of defense-related genes,such as pattern-recognition receptors(BAK1 and FLS2),ethylene-related genes(EIN2,EIN3,and ERF),and reactive oxygen species-related genes(RBOH),which thus inhibits the penetration of haustorium and confers plant resistance to dodder.3.Field evaluation of CuRe1-overexpressed alfalfa against dodder parasitism.Three consecutive-year field trials showed that CuRe1-overexpressed alfalfa exhibited stronger resistance against dodder parasitism than the control plants.We also found that the dry weight of CuRe1-overexpressed alfalfa increased by 22.9%(2019),23.6%(2020),and 21.8%(2021)than the control alfalfa plants,respectively.The CuRe1-overexpressed alfalfa plants exhibited a stronger capacity for photosynthesis,antioxidant defense,and osmotic adjustment,resulting in more accumulation of dry matter and higher forage quality.An artificial hybridization experiment revealed CuRe1 gene could be stably inherited in alfalfa generations.