CRY2-mediated Regulation Analysis Of ICS1 Expression By TCP8 In Arabidopsis

Successful disease development requires that pathogen,susceptible host,and favorable environmental conditions come together at the same time.Plant defense against pathogens is significantly influenced by environmental factors such as temperature,humidity,light as well as circadian rhythm,among which light-dependence of plant immunity is mediated by photosynthesis as well as photoreceptor signaling.With regard to photoreceptors,initially,a link between phytochrome-mediated signaling and the defense response was proposed,as phy Aphy B double mutant was reported to display a reduced HR and an increased susceptibility to the avirulent strain Pst DC3000 avr Rpt2.Studies on the CRY-mediated regulation of plant immunity remain on the CRY2-mediated degradation of R protein HRT in a ubiquitination dependent manner.In this study,TCP8 was screened out by a yeast two-hybrid system using CRY2 as a prey protein.TCP8 regulates the expression of ICS1 by binding to the promoter of ICS1,promoting systemic acquired resistance in plants.Therefore we did some research to confirm whether the regulation of ICS1 by TCP8 depends on CRY2.1.TCP8 was screened out using CRY2 as a prey protein.The blue-light specific interaction between TCP8 and CRY2 further confirmed by Histidine auxotrophy assays and CPRG ?-galactosidase activity assay.The interaction between TCP8 and CRY2 was verified again by Co-IP of these two proteins co-expressed in HEK293 T cells without blue-light specificity.Co-IP assay was performed again in plant,which further validated the interaction of these two proteins,.2.Leaf discs was harvested 3 days after Pst DC3000 inoculation,and then it was used for analysing disease symptoms and Pst DC3000 growth in different genotypes.Comparing to Col4,cry2 and cry1cry2 exhibited an increased susceptibility to Pst DC3000 inoculation,and GFP-CRY2 showed a reduced susceptibility to Pst DC3000 because of reduced growth of bacteria,which suggests CRY2 plays a key role in systematic acquired resistance in plants.Leaf discs was harvested at indicated time points after Pst DC3000 inoculation to measure the expression of ICS1.The extent of increased expression of ICS1 in blue light was larger than that in dark.3.By comparing the expression of ICS1 in four Arabidopsis genotypes of ACT2::GFP-TCP8/Col-4,ACT2::GFP-TCP8/cry2,Col4 and cry2 in two different conditions of dark and blue light as well as performing luciferase activity assay in HEK293 T cells,the regulation of ICS1 by TCP8 dependent CRY2 was validated.However,this regulation may not depend on blue light and may depend on LWD(LIGHTREGULATED WD).