Study On Programmed Cell Death In Euneka Lemon Induced By Citrus Yellow Vein Clearing Virus

Citrus yellow vein disease,caused by Citrus yellow vein clearing virus(CYVCV),seriously threatens the production of lemon and lime.It causes leaf veins and yellowing.In 2009,CYVCV was observed on Eureka lemon in Yunnan,and then soon it was found in Chongqing and Sichuan,especially in Anyue,Sichuan.Previous studies indicated that programmed cell death(PCD)was involved in plant disease resistance.Hypersensitive reaction(HR),caused by the interaction between plants and pathogens,is one of the important response of plant defense against pathogen invasion.It is also a manifestation of PCD,which is related to plant disease resistance.This study was aimed at exploring the mechanism of PCD induced by CYVCV based on the relationship between CYVCV contents and PCD symptoms detected by TUNEL,microscopy and agarose gel electrophoresis,as well as the expression analysis of PCD-related genes and enzymes at the different stages.The main results are as follows:1.CYVCV were found in the phloem of the main vein,secretory cavity and parenchyma cells in Euneka lemon leaves by DTBIA and fluorescence immunoassay.2.The results of TUNEL detection showed that positive signals of PCD appeared more and more in leaves of Euneka lemon with the increase of infection time and the quantity of CYVCV.However,the quantity of CYVCV and PCD positive signals of PCD both decreased in the late infection period.The results suggested that CYVCV could induce the production of PCD in Euneka lemon,and more PCD symptoms were detected with the increase of CYVCV contents.3.PCD appeared in roots,stems and leaves after CYVCV infection.The root positive reaction was the strongest,followed by the stems and leaves.It positively correlated with the quantity of CYVCV.4.The ultrastructural changes induced by CYVCV showed :(1)The increase of starch grains,more osmium particles,chloroplast swelling,deformation and disintegration in the chloroplast;(2)Outer membrane dissolution,crust blur and disintegration in some mitochondria;(3)Chromatin condensation and marginalization,nuclear membrane dissolution and high concentration.The results also demonstrated that PCD could be induced by CYVCV in Euneka lemon.5.The relative expression levels of CsCysp,Cit-Dad1-1 and MCA1 were detected by qRT-PCR.CsCysp and Cit-Dad1-1 genes were down-regulated to inhibit the production of PCD.The MCA1 gene expression increased might be related to the increase of caspase activity to promote the production of PCD.6.The activity of Caspase-3 protease increased with the increase of infection time by CYVCV.The content of malondialdehyde decreased first and then increased.It was higher than the control group,which indicated that the membrane lipid peroxidation was increased.The activity of SOD,CAT and POD decreased to weaken the antioxidant capacity of cells at the early infection stage.It induced the accumulation of ROS and the production of PCD.The activity of antioxidant enzymes increased to remove ROS and to inhibit the production of PCD at the late infection stage.