ATL99s Role in Plant Defense Figure 2. Influence of SA-, JA- and ET-mediated pathways on ATL9 expression measured by qRT-PCR. A) Induction of ATL9 30 minutes after chitin treatment in Col-0 wild-type plants and in the ein2-5, jar1 and sid2-1 mutants. B) Induction of ATL9 (black), PR1 (light gray) and PDF1.2 (dark gray) in Col-0 plants after treatment with chitin (CSC), 50 mM ACC, 5 mM JA or 0.5 mM SA. Data represent the ratio between mock and treated plants of three independent biological samples. The inset shows relative expression of ATL9 transcript compared to the actin control (RE/Actin). doi:10.1371/journal.pone.0014426.g002 compared to wild-type plants. AtrbohC plants are also more susceptible to the powdery mildew pathogen G. cichoracearum (data not shown). These data raised the possibility that the induction of ATL9 by chitin might be dependent on NADPH oxidase activity. To test this hypothesis we treated Col-0 plants and three mutants impaired in NADPH activity (AtrbohD, AtrbohF, AtrbohD/F) with chitin and analyzed the transcript levels of ATL9 by qRT-PCR (Figure 4). The MAPK3 gene was used as a control since it is known to be induced by chitin [29]. Though ATL9 was induced by chitin in Col-0 plants, its induction was significantly reduced in the three Atrboh mutants (Figure 4). ATL9 was still expressed in untreated Atrboh mutants but at half of that observed in untreated wild-type plants (inset Figure 4). These data indicate that basal expression levels of ATL9 are dependent on AtrbohD and F.. A similar pattern of reduced induction and reduced basal expression was observed for the MAPK3 gene in all the lines tested (Figure 4 and inset). These results indicate that both basal transcription levels of ATL9 as well as the induction of ATL9 by chitin depend on NADPH oxidase activity. These results are in line with previous NASC array data showing that ATL9 transcription levels are impaired in the AtrbohC mutant. ATL9 is induced by G. cichoracearum and has a complex pattern of expression post-inoculation Since atl9 mutants are more susceptible to G. cichoracearum (Figure 3A) than wild-type plants and ATL9 basal expression and induction by chitin is dependent on NADPH oxidases (Figure 4 PLoS ONE | www.plosone.org 4 December 2010 | Volume 5 | Issue 12 | e14426