Research On The Mechanisms Of CaWRKY20 And CaWRKY50 Under Colletotrichum Scovillei Infection And QTLs Mapping Of Anthracnose Resistance In Capsicum Annuum

Pepper（Capsicum annuum L.）is one of the most important economic crops around the world,with high nutritional value.Although modern breeding and cultivation techniques have greatly improved,the harm of pathogens remains a key limiting factor in the development of the pepper industry.Among them,anthracnose caused by Colletotrichum spp.has greatly affected the yield and the high quality of pepper,which is one of the most destructive fungal diseases in the pre-and post-harvest stages of pepper.Colletotrichum scovillei is considered a relatively common and destructive of anthracnose.In order to study the disease resistance mechanism to anthracnose,we screened for disease resistance related genes through forward genetics,and verified the important regulatory function of WRKY transcription factor（TF）in disease resistance based on reverse genetics.Quantitative trait locus（QTL）and map-based cloning technology provide a rapid and effective ways to screen for the resistance genes.In addition,WRKY TF regulates the response of plants to various stress and plays a crucial role in disease resistance and stress of plants.However,the function of WRKY TF in pepper resistance to anthracnose is still unclear.In this study,two CaWRKY genes responding to pepper anthracnose,CaWRKY20 and CaWRKY50,were identified by analyzing the transcriptome data in the previous study of our research group and using virus-induced gene silencing（VIGS）technology.We also studied the regulatory mechanism of these two genes under C.scovillei infection.At the same time,the QTLs related anthracnose resistance was mapped in pepper.The main research results are as follows:1.By analyzing the transcriptome data based on the previous studies in our group,seven differentially expressed CaWRKYs genes were screened.These CaWRKYs genes were silenced by VIGS technology on detached pepper fruits and then inoculated them with C.scovillei to identify the disease resistance of pepper fruits.The results showed that the lesion diameter of CaWRKY20 and CaWRKY50-silenced fruits were significantly smaller than the control fruits.While the lesion diameter of other genes silenced fruits were not significantly different from the control.Therefore,it is speculated that CaWRKY20 and CaWRKY50 may be involved in the defense response of pepper against C.scovillei infection.2.CaWRKY20 negatively regulated the resistance of pepper to C.scovillei.We treated detached pepper fruits with salicylic acid（SA）.The results showed that CaWRKY20 can be induced by SA.The CaWRKY20 protein is localized in the nucleus and has transcriptional activation activity.CaWRKY20 transiently expressed on detached pepper fruits or CaWRKY20stable overexpression in tomato increased the content of malondialdehyde（MDA）and H₂O₂,reduced the scavenging ability of reactive oxygen species（ROS）,and inhibited the expression of SA defense-related genes（CaPR1,CaPR10 and CaSAR8.2）and ROS scavenging genes（CaCAT,CaPOD and CaSOD）resulted in decreasing the resistance to C.scovillei.While silencing of CaWRKY20 on detached pepper fruits enhanced the resistance to C.scovillei.Additionally,it was found that CaWRKY20 interacts with two E3 ubiquitin ligase proteins CaMIEL1 and CaCHYR1 by using yeast two-hybrid（Y2H）technique.Through luciferase complementation imaging（LCI）and bimolecular fluorescence complementation（Bi FC）technique,it was demonstrated that CaWRKY20 interacts with CaMIEL1 and CaCHYR1 in the nucleus.Meanwhile,CaWRKY20 inhibits the expression of the SA defense genes CaACD6 and CaSARD1 by directly binds to the W-box（TTGACT/C）element in the promoters of them.These results indicated that CaWRKY20 plays a negative regulatory role in the defense response to C.scovillei in pepper,which is regulated by SA signaling pathway and antioxidant defense system.3.CaWRKY50 negatively regulated the resistance of pepper to C.scovillei.CaWRKY50can be induced by SA treatment.The CaWRKY50 protein is localized in the nucleus but has no transcriptional activation activity.Silencing of CaWRKY50 on detached pepper fruits enhanced the resistance to C.scovillei.Meanwhile,CaWRKY50 transiently expressed on detached pepper fruits or CaWRKY50 stable overexpression in tomato increased the content of MDA and H₂O₂,reduced the scavenging ability of ROS,and inhibited the expression of SA defense-related genes（CaPR1,CaPR10 and CaSAR8.2）and ROS scavenging genes（CaCAT,CaPOD and CaSOD）resulted in decreasing the resistance to C.scovillei.Through the Y2H,LCI and Bi FC technique,it was demonstrated that CaWRKY50 could interact with CaWRKY42 and CaMIEL1 in the nucleus,which may be involved in pepper defense response against C.scovillei.Besides,CaWRKY50 could directly binds to the W-box（TTGACT/C）in the promoters of SA defense genes CaEDS1 and CaSAMT1 to inhibits their activity.These results indicated that CaWRKY50 plays a negative regulatory role in the defense response to C.scovillei in pepper through SA and ROS signaling pathways.4.Two lipoxygenase genes related to anthracnose,CaLOX6 and CaLOX7,were identified by QTL mapping using segregation populations F₂,F_2:3 and F_3:4 constructed from crossing resistant parent R24 with susceptible parent R25.The genetic analysis showed that the pepper resistance to C.scovillei was a dominant inheritance controlled by multiple genes.The genetic linkage map was constructed using 186 F₂ populations.This linkage map covers a length of about 1614.02 c M.The QTL locus q AR1.1 for anthracnose resistance in pepper was detected between 01g088-01g085 markers on chromosome 01 using F₂ population and F_2:3 families.Then F_3:4 families was used to narrow the q AR1.1 locus to markers 01g282-01g256,with a physical distance of 1.56 Mb and 18 genes within the interval.Based on functional annotation information of pepper gene and q RT-PCR analysis,it is speculated that two lipoxygenase genes CaLOX6（Capana01g001574）and CaLOX7（Capana01g001578）may be involved in the defense response to C.scovillei in pepper through jasmonic acid（JA）signaling pathway.In summary,this study revealed that CaWRKY20 and CaWRKY50 played a negative regulatory role in the defense response to C.scovillei in pepper by regulating SA and ROS signaling pathways.It was predicted that CaLOX6 and CaLOX7 might be involved in the defense response to C.scovillei in pepper by QTLs mapping associated with anthracnose resistance.These results enriched the regulatory network of pepper WRKYs and LOXs participating in disease resistance to C.scovillei,laying the foundation for breeding pepper resistance to anthracnose.