Mechanisms Of Negative Regulation Of Plant Resistance Against Phytophthora Parasitica By Transcription Factor ERF019

Oomycetes,including Phytophthora,downy mildew,and Pythium,pose a great threat to sustainable crop production.Due to the evolutionary differences between fungi and oomycetes,many fungicides are ineffective against Phytophthora pathogens.One more notable feature of oomycetes in causing crop diseases is its strong plasticity in virulence,being capable of deriving virulent progenies to defeat host resistance,leading to frequent loss of crop genotype-specific disease resistance.Thus,elucidating the molecular mechanism of plant resistance to Phytophthora will be useful for revealing the mechanism of plant-oomycete interactions and for developing novel disease control strategies to ensure food security in the world.In this study,we employed the model oomycete pathosystem Arabidopsis thaliana-Phytophthora parasitica to understand compatible plant-pathogen interaction.We focused on one T-DNA insertion mutant 267-31,which contains two adjacent T-DNA insertion sites in the promoter region of ETHYLENE RESPONSIVE FACTOR 19(ERF019).We used genetic methods to prove that ERF019 negatively regulates plant resistance,and then used molecular biological and biochemical methods to reveal the regulation of ERF019 in plant resistance to P.parasitica.In addition,we also analyzed the immune function of the ortholog of ERF019 in Nicotiana benthamiana to explore the possibility of using ERF019 in crop breeding for enhanced disease resistance.The main results are as follows:1.Generated CRISPR/Cas9-edited erf019 mutants and ERF019 overexpression plants.Pathogenicity assays showed that the CRISPR/Cas9-edited erf019 mutants exhibited enhanced resistance to P.parasitica,while ERF019-overexpression lines plants showed increased susceptibility to P.parasitica,indicating that ERF019 is a negative regulator of plant resistance to P.parasitica.2.Determined the expression pattern of ERF019 in response to P.parasitica infection and hormone treatment by q RT-PCR assay.The results showed that the expression of ERF019 was induced with high levels at the early stage,3 h post inoculation with P.parasitica zoospores.The expression of ERF019 was induced by SA and Me JA,but not ET,and peaked at 1h post treatment.Furthermore,the expression of defense-related genes was altered in the erf019 mutants.Compared to the wild type Col-0,the expression of SA biosynthesis related gene ICS1,SA signaling pathway marker gene PR1,JA biosynthesis related gene LOX2,and JA signaling pathway marker genes PDF1.2 and VSP2 were up-regulated in multiple erf019 mutants,while the expression of ET biosynthesis related genes ACS2 and ACS6,ET signaling pathway marker genes EIN2 and ERF6 appeared similar between erf019 mutants and the wild type.These results indicate that ERF019 may play a role in plant resistance to P.parasitica by participating in the SA and JA signaling pathways.3.Determined that the nuclear localization of ERF019 is required for its immune function to P.parasitica.Transient overexpression assays showed that ERF019-GFP protein promoted P.parasitica colonization in N.benthamiana,while the ERF019-GFP-nuclear export sequence(NES)fusion protein,but not ERF019-GFP-mutant NES(nes)fusion protein,lost its ability to enhance P.parasitica colonization.In addition,we employed the dexamethasone(DEX)-induced nuclear relocation system and prepared transgenic plants that express ERF019-rat glucocorticoid receptor(GR)fusion protein.Pathogenicity assays showed that the nuclear re-localization of ERF019-GR induced by DEX promoted P.parasitica growth.4.Determined that ERF019 negatively regulates PAMP-triggered immunity(PTI)response.Immunoblotting experiments were performed to detect phosphorylated MAP kinase upon flg22 treatment in ERF019-OE lines and multiple erf019 mutants.The results showed that PAMP-triggered activation of MAPKs was much stronger in erf019 mutants and was compromised in ERF019-OE lines.Furthermore,flg22-induced accumulation of hydrogen peroxide and ROS burst were impaired in ERF019-OE plants.In addition,transient overexpression of ERF019 specifically suppressed Phytophthora elicitor INF1-triggered cell death in N.benthamiana.These results suggest that ERF019 negatively regulates PAMP-triggered immunity.5.Determined the conserved immune function of ERF019 across distant plant species.Explored the potential function of Nb19,the homologous gene of At ERF019 in N.benthamiana,in plant immunity.Nb19 was induced by P.parasitica infection and flg22 treatment.Overexpression of Nb19 not only suppressed the induction of FRK and WRKY33 after flg22 treatment,but also inhibited ROS burst,and promoted the colonization of P.parasitica in N.benthamiana.In addition,silencing of Nb19 had no significant effect on plant growth,but accelerated INF1-and Bax-triggered cell death.In conclusion,our results show that ERF019 negatively regulates plant resistance to P.parasitica by suppressing PAMP-triggered immunity.For further work,identification of ERF019 interacting protein by IP-MS/MS and ERF019 targeted genes using Ch IP-Seq technology will provide further insights into the mechanism of ERF019-mediated plant immunity to P.parasitica.Considering that erf019 mutants showed normal growth phenotype while exhibited enhanced resistance to P.parasitica,functional analysis of its homologous genes in crops will have great potential to facilitate breeding for durable disease resistance.