An Improved Transformation System For Phytophthora Cinnamomi Using Green Fluorescent Protein And Function Analysis Of RxLR Effector PcAvh87

Phytophthora cinnamoni has a wide range of hosts and is capable of infecting more than5,000 plants.P.cinnamoni causes forest loss in Europe,Americas and Asia and is considered a threat to biodiversity.P.cinnamoni can cause root rot in many agricultural and forestry crops in China.Current studies have found that Phytospora secretes a large number of effector proteins to attack the immune system of plants during the infection of the host,among which there is a large class of protein family called RXLR effector,which contains a conserved Arg-X-Lys-Arg（RXLR）sequence after the signal peptide.There are 266 RXLR effectors encoded in the genome of P.cinnamoni.However,there are few reports on effector proteins of P.cinnamoni,and the molecular mechanism of how effector proteins regulate host immunity is very little known.Based on previous studies,an effector PcAvh87 that can inhibit INF1 and Bax induced cell deathwas obtained,which indicated that PcAvh87 may play a certain role in the interaction between Phytophthae and plants.In order to clarify the role of PcAvh87 in the pathogenesis of P.cinnamoni,a stable and efficient PEG/Ca Cl₂-mediated genetic transformationsystem was established using green fluorescent protein（GFP）.Furthermore,PcAvh87 mutants were obtained by PEG/Ca Cl₂mediated protoplast transformation using CRISPR/Cas9-mediated gene editing,to explore the function and host interaction mechanism of effector PcAvh87.This studyaims to elucidate the novel pathogenicity mechanisms of Phytophthora pathogens,and will provide scientific basis for establishment of novel control strategies against Phytophthora pathogens.The main research results are as follows:1.Phytophthora cinnamomi is a destructive pathogen causing root rot and dieback diseases on hundreds of economically and ecologically important plant species.Effective transformation systems predispose modifications of candidate genes to understand the pathogenesis of P.cinnamomi.A previous study reported a polyethylene glycol and calcium dichloride（PEG/Ca Cl₂）mediated protoplast transformation method of P.cinnamomi.However,the virulence of the transformants was compromised.In this study,we selected ATCC 15400 as the suitable wild-type isolate for PEG/Ca Cl₂transformation using the green fluorescent protein after screening 11 P.cinnamomi isolates.Three transformants,namely Pc GFP-1,Pc GFP-3,and Pc GFP-5 consistently displayed a green fluorescence in their hyphae,chlamydospores,and sporangia.The randomly selected transformant Pc GFP-1 was as virulent as the wild-type isolate in causing hypocotyl lesions of lupins.Fluorescent hyphae and haustoria were observed intracellularly and intercellularly in lupin tissues inoculated with Pc GFP-1 zoospores.The potential application of this improved transformation system for functional genomics studies of P.cinnamomi is discussed.2.In this study,analyses of gene expression patterns revealed that a Rx LR gene,PcAvh87,was highly induced in the early stage of infection.Using transient expression assays,we found that the efffector PcAvh87 fully suppressed programmed cell death of Nicotiana benthamiana leaves elicited by bothpro-apoptotic protein BAX and elicitin protein INF1.Sequences of the PcAvh87 gene of eight P.cinnamomi isolates collected from four countries and five host plants are identical,suggesting that its role is essential and non-redundant during infection.Homologues of PcAvh87 were identified in other oomycetes including P.infestans,P.sojae,P.capsici,P.parasitica,P.kernoviae and P.palmivora,but not in fungi or other organisms.We also discovered that PcAvh87 could localized to the plant cell nucleus.Functional analysis revealed that PcAvh87 significantly promoted the infection of Phytophthora nicotianae.PcAvh87 mutants were obtained by PEG/Ca Cl₂mediated protoplast transformation using CRISPR/Cas9-mediated gene editing.PcAvh87 mutant severely impairs the virulence,indicating that PcAvh87 is an important virulence factor.PcAvh87 is involved in the regulation of hyphal growth,sporangium development and zoospore release,and PcAvh87mutants were significantly more sensitive to oxidative stress.3.High-throughput RNA-seq technology was used to carry out comparative transcriptome analysis of wild-type strain ATCC 15400 and PcAvh87 knockout mutant strain T5 at the mycelial and zoosporal stages,and a total data volume of 84.62 G was obtained by sequencing.Through comparison of PHI database,339 genes were differentially expressed in mycelium stage,508 genes in zoosporial stage,320 genes were significantly up-regulated,and 188 genes were significantly down-regulated.These PHI genes mainly included plant cell wall degrading enzyme,NLP,activator and RXLR effector.The results showed that due to the knockout of the effector PcAvh87,the expression of about 1/10 genes in the genome of P.cinnamoni was changed,and the expression of most genes was down-regulated.There were 85 cell wall degrading enzymes and 36 effectors,including 13 excitons,7 NLP and 16 Rx LR effectors.4.Yeast hybridization was used to screen the interacting proteins of effector PcAvh87 in lupine to analyze the influencing factors of pathogen infection strategy.The results showed that the titer of the lupin yeast hybrid library constructed in this study was 7.38×10⁶CFU/m L.A total of 68 interacting proteins were screened in the lupin yeast hybrid library,and the functions of the screened proteins involved in stress response,immune regulation and plant development.The two strongly interacting proteins were Avh87-113 and Avh87-123.Avh87-113 is a homologous protein of NHL1.NHL1（NDR1/HN1-like）protein plays a key role in various plant defense responses.NHL1（NDR1/H1N1-like）was extracted from the Arabidopsis thaliana genome database and correlated with Arabidopsis thaliana NDRL（non-race-specific disease resistance 1）and tobacco H1N1（Harpin induced）Gene1)is a homologous gene that is necessary for the Resistance gene products of Arabidopsis thaliana to bacteria and bacterial pathogens,and playsan important role in the defense and immune responses of a variety of plants.The functional annotation of Avh87-123 is a small ubiquitin-like modifier（SUMO-1）,which plays an important role in plant development,regulation of hormone levels,disease resistance and response to adversity.Mining the host interaction protein of PcAvh87 is helpful to understand the pathogenic mechanism of effector PcAvh87.