ATL99s Role in Plant Defense Figure 8. Microarray analysis of atl9 mutant after chitin treatment. A) Hierarchical cluster of ratio values (relative to the water control treatment) of 16,530 genes analyzed in Col-0 wild-type and atl9 plants treated with crab shell chitin (CSC) for 30 minutes. Each gene is represented by a single row and each column represents an individual treatment. Red represents up-regulated genes; blue, down-regulated genes; and yellow, genes with no change in expression. Groups of genes expressed differentially are delineated with rectangles. Three genes (black arrows) were changing due to interactions between treatment and genotype and one gene (red arrow) was genotype interaction specific. B) Venn diagram representation of results from hierarchical clustering. A total of 4,375 genes were differentially expressed between genotypes and treatments. The statistic used for clustering was two-way ANOVA. Genotype and treatment groups were analyzed using a p-value of 0.5 with p-value .0.5 = not significant; p-value ,0.5 = significant. doi:10.1371/journal.pone.0014426.g008 induced by chitin [23,26]. EL5, like ATL9, is an E3 ubiquitin ligase and is hypothesized to play a role in defense responses through protein turnover via the UPS [22]. Additionally, the ACRE-132 gene, an ATL protein from tobacco has been shown to be induced during the defense response mediated by the interaction between Avr9 and Cf9 in the response to the fungal pathogen Cladosporium fulvum [46]. ATL9 is unique among ATL family members that have been characterized since it contains a PEST (Pro-Glu-Ser-Thr) domain. PEST domains are common in proteins that are rapidly degraded in the cell [47] and are suggested to serve as proteolytic signals [48] involved in ubiquitination and internalization of proteins. While few studies in plants have examined the role of PEST domains and the UPS in defense, there are numerous studies in mammals describing PEST domains and their role in defense against cellular pathogens [49]. The presence of a PEST domain in the ATL9 protein may imply that there are multiple ways to control its expression and that ATL9 may play a critical role in plant defense similar to that of the Mcl family of genes in humans which are necessary for cell survival in the immune system [50]. In plants, the tomato Ve gene contains a PEST domain that is necessary for defense against Verticillim species [51]. Our data show that ATL9 is necessary for resistance against G. cichoracearum (Figure 3A). ATL9 transcription levels appear to be tightly regulated; after chitin treatment the gene is induced within 30 minutes [6] and in plants infected with G. cichoracearum ATL9 expression can be detected in less than one hour by qRT-PCR (Figure 5). We hypothesize that ATL9 expression is also down- regulated quickly (,1 hour) with ATL9 binding its target for elimination via the UPS followed by rapid ATL9 degradation via its PEST domain. Over-expression of ATL9 produced plants that were more resistant than wild-type (Figure 3A) suggesting that the gene is degraded rapidly under normal conditions in wild type plants post-infection. Further experiments with deletion constructs in the PEST domain of ATL9 will be useful in characterizing ATL99s regulation and in confirming our hypothesis regarding its degradation. ATL9 appears to be involved in ER stress responses and ERAD during innate immunity In 2003, Takemoto et al. showed cytoplasmic aggregation and accumulation of ER and Golgi bodies around the sites of fungal penetration [52]. These cellular rearrangements suggest that the production and secretion of plant materials are activated around PLoS ONE | www.plosone.org 9 December 2010 | Volume 5 | Issue 12 | e14426