Histological And Cytological Research On Infection Process Of Two Effector Genes Mutants From Valsa Mali

Apple Valsa canker caused by the ascomycete Valsa mali is widely distributed in China.This fungus can result in serious damage and even destructi on of the orchard.It was proved that the effector protein secreted by V.mali significantly promoted the pathogen infection.Therefore,studying the interaction process between the host and the pathogen can provide more evidence for the analysis of the function of effector proteins from the perspective of histology and cytology,which is of great significance to reveal the pathogenic mechanism V.mali.In previous studies,a cell death suppressor?Vm EP1?and a cell death elicitor?Vm E02?were identified from V.mali candidate effector proteins.The virulnce of Vm EP1 knockout mutant??EP1?decreased by about 50%and it was proved to be an effector gene.Knocking out of Vm E02 did not show visible alteration to the virulence of V.mali significantly,while Vm E02 overexpression mutants?OE-E02?increased pathogen virulence by 14.28%,indicating it is an effector gene as well.In order to explore the effects of these two effector proteins in host tissue and cell morphology changes under V.mali infection,the histological and cytological studies of two effect gene mutants infecting apple hosts were conducted.The results and conclusions were as follows:1. The 3D models of the interaction between V.mali and apple host were constructed based on Microtomy-CLSM technology.The author observed the spatial structure of the colonization of V.mali in apple twigs and leaf tissues,the spatial structure of the highly hydrolyzed host tissues cross-linked with V.mali and the complete fiber structure.On these bases,the 3D models of interaction between V.mali and apple host were constructed to analyze the sophisticated structure of the combination of V.mali and the host,which provides more evidence and optimized technical support for subsequent pathogenic research.2. Host damage caused by these three strains ranges:OE-E02 strain>03-8 wild type strain>?EP1 strain;the mutants strains triggered thickening of host cell wall this kind of immune response faster.Histological sections at 1 to 5 day?s?post inoculation?dpi?of the three strains showed all three strains caused a slight to severe degradation of the host tissues.At 5 dpi,the OE-E02 strain caused complete hydrolysis of host tissue,the 03-8 wild type strain caused extensive hydrolysis of host tissues and the?EP1 strain caused partial hydrolysis of host tissues.Cytological studies showed that host cells collected from the diseased tissues showed the phenomena of plasmolysis,cytoplasm condensed,cell wall degradation,vesiculation,disintegration of various organelles,cell hydrolysis and necrosis.Under artificial inoculation conditions,host's immune response under 03-8 strain treatment such as thickening of the host cell wall was found at 1.0 cm/48 h away from the inoculation center.?EP1 strain at 0.5 cm/24 h and OE-E02 strain at 0.5 cm/12 h and 1.0 cm/12 h caused the thickening of the host cell wall faster than those of 03-8 treatment.3. The infection of?EP1 and OE-E02 strain triggered the response mechanism of reactive oxygen species?ROS?in apple host faster,accumulating more ROS than that of 03-8strain.To further study the host's response to?EP1 and OE-E02 strains,cytochemical localization of reactive oxygen species?ROS?during the infection process was carried out.The Mn²⁺/DAB precipitation method and the Ce Cl₃precipitation method were used to localize O₂^-and H₂O₂in apple leaf cells inoculated with three strains respectively for 12 h.It was found that the host cell membrane system,the inside and outside of the cell wall,the intercellular space and the hyphae all appeared a large number of continuous black precipitates?O₂^-,H₂O₂?under?EP1 and OE-E02 treatment;under the 03-8 treatment,only a partial black O₂^-precipitation appeared on the host cell membrane,however,no O₂^-precipitation was observed around the hyphae,and a small amount of granular black H₂O₂precipitation appeared on both the inside and outside of the host cell,no H₂O₂precipitation was observed around the hyphae.4. ?EP1 and OE-E02 strain triggered a faster and stronger immune response of the host.In order to investigate the changes of the host's immune response during the infection of V.mali,the expression patterns of apple host defense genes were analyzed.Apple twigs inoculated with V.mali 03-8 wild type strain,?EP1 strain and OE-E02 strain respectively were sampled at 0,12,24,36 and 48 hour?s?post inoculation?hpi?,and it showed that the host Md PDF1,Md NPR1,Md PR1 and Md PR2 gene all had a significant up-regulation trend,and the variation trend of Md NPR1 was similar.The peak expression of host defense genes under?EP1,OE-E02 treatment was greatly increased and the time of Md PDF1,Md PR1 and Md PR2 gene arrived earlier than 03-8 treatment.Under the 03-8 treatment,the peaks of Md PDF1,Md NPR1,Md PR1 and Md PR2 gene appeared at 48 h,36 h,48 h and 48 h respectively;the peaks of the genes under?EP1 treatment all appeared at 36 h,and the peak values of the genes were 2.88,1.59,9.14 and 2.42 fold higher than those of 03-8 treatment respectively;the peaks of the genes under OE-E02 treatment all appeared at 24 h,36 h,36 h and 24 h respectively,and the peak values of the genes were 1.36,1.30,4.03 and 2.75 fold higher than those of 03-8 treatment.Based on the results of the above studies,it was found that the Valsa mali?EP1 and OE-E02 strain can cause a stronger immune response on apple host.We confirmed that Vm EP1 is an important gene which suppresses the host's immune response,and Vm E02 is an important elicitorgene which triggers the host's immune response.This study established the significant basis for further exploration of the interaction mechanism between the two effector proteins and apple host.