Functional Analysis Of Drosophila Inhibitor Of Apoptosis Like Proteins And MA3 Domain-containing Topoisomerases In Programmed Cell Death In Arabidopsis

Programmed cell death(PCD) is critical for normal growth/development,maintenance of tissue homeostasis and for defense responses in plants,regulating precise death of unwanted cells.In plants,the existence of PCD has been firmly established,and a number of plant proteins isolated recently have been assigned a PCD role.However,the molecular and genetic basis of PCD in plants remains largely unclear yet.In the present study two Arabidopsis homologues:AtDAL1 and AtDAL2,showing similarity to Drosophila inhibitor of apoptosis(DIAP) were identified through homology searching using DIAP as queries and performed detailed studies using T-DNA insertion lines to analyze the possible functions of these two genes in PCD induced by pathogen infection or chemical inducer.Meanwhile, possible involvement of a small subgroup of MA3 domain-containing topoisomerases in PCD of Arabidopsis was also investigated.DIAP1,a functional anti-apoptotic protein in Drosophila melanogaster,not only controls cell death,but also influences signal transduction pathways for differentiation, protein turnover and progression through the cell cycle.Two Arabidopsis genes,DAL1 and DAL2(for Drosophila inhibitor of apoptosis like 1 and 2),encoding zinc finger proteins, were identified.The DAL1 and DAL2 proteins show similarity to DIAP1 and contain a RING domain at C-terminus.RT-PCR analysis showed that expression of DAL1 and DAL2 were induced in Arabidopsis plants after inoculation with virulent and avirulent strains of Pseudomonas syrinage pv.tomato(Pst) DC3000.The expression of DAL1 and DAL2 was also induced in Arabidopsis plants after infiltration with Fumonisin B1(FB1):a PCD inducing fungal toxin.To study the function of DAL1 and DAL2 in PCD,T-DNA insertion lines dal1-1,dal1-2,dal2-1 and dal2-2(two lines of each gene) were identified from genotype screening.RT-PCR analysis indicated that transcript of the DAL1 or DAL2 genes were undetected in the dal mutant plants,suggesting that dal1-1,dal1-2,dal2-1 and dal2-2 are knockout mutants of the DAL genes.The phenotypes of the dal plants after inoculation with Pst DC3000 virulent strain showed similar and/or slightly higher disease symptoms than the wild type.When inoculated with avirulent strain Pst DC3000-AvrRpml,all four dal mutants displayed higher levels of disease than the wild type plants.Although inoculation with the virulent and avirulent Pst DC3000 strains resulted in induction of PR-1 genes,no significant difference in PR-1 expression was observed between wild-type and the dal mutant plants.After inoculation with the avirulent strain of Pst DC3000,a significant accumulation of superoxide anion and prominent cell death were observed in dal mutant plants.All dal1-l,dal1-2,dal2-1 and dal2-2 mutant plants exhibited accelerated progression of cell death upon infiltration of leaf tissues with FB1 and displayed significant cell death as well as accumulation of superoxide anion over the wild type plants.Appearance of autofluorescent compounds and callose deposition were significantly higher than those in the wild-type plants.The dal mutant plants showed increased susceptibility to Alternaria brassicicola but did not alter the disease phenotype against Botrytis cinerea.Ectopic expression of DAL1 and DAL2 in yeast did not inhibit H₂O₂-induced cell death.These data suggest that DAL1 and DAL2 represent two novel regulators of PCD in Arabidopsis.In animal system,it has been shown that aberrant expression of genes encoding topoisomerases is often associated with the induction of apoptosis.Deregulated expression of topoisomeraseⅡαis associated with the commitment of apoptotic cell death.In this study, a small subgroup of MA3 domain-containing topoisomerases was identified from Arabidopsis and it was designated as TOP1-4.RT-PCR analysis showed that expression of TOP1 and TOP2 were induced in Arabidopsis plants after inoculation with the virulent and avirulent strains of Pst DC3000 and that the expression level was significantly up-regulated. To study the function of TOP1-4,T-DNA insertion lines top1,top2,top3 and top4 were identified from genotype screening and confirmed by RT-PCR analysis.The top1,top2, top3 and top4 are knockout mutants of the TOP genes.The top mutant plants showed lesser disease symptoms than the wild type after the inoculation with Pst DC3000 virulent strain. However,when inoculated with Pst DC3000 avirulent strain all four top mutants displayed higher levels of disease than the wild type plants.When the plants were inoculated with a necrotrophic pathogen Alternaria brassicicola,top3 plants did not show any altered response compared to the wild type plants,top4 showed reduced response and top1 and top2 exhibited significantly increased disease symptoms compared to the wild type.The top1, top2 and top3 mutant plants induced appearance of autofluorescent compounds and callose deposition in both vascular and non-vascular tissues while in the wild type it was restricted to the vascular tissues.The top1 and top2 plants showed altered resistance to different ABA concentrations displaying stunted plants.These results indicate this small subgroup of MA3 domain-containing topoisomerase involves in PCD and defense response against pathogen infection.