ATL99s Role in Plant Defense Figure 1. ATL9 structure and sequence alignment between ATL family members. A) Schematic diagram of ATL9 protein structure. The protein has an N terminal region (cytosolic), containing a signal peptide (gray); two transmembrane domains (Tm1 and Tm2, in black); and a C3HC3 RING Zinc-finger domain (dark gray), a PEST sequence and a coiled coil domain that extends into the ER lumen (white). B) Alignment of ATL9 RING Zinc-finger domain amino acid sequence with the other ATL family members implicated in defense responses in plants. Asterisks indicate conserved cysteines; conserved amino acids are indicated in black, residues conserved in more than an 87.5% are highlighted in gray. The consensus sequence for this group of RING zinc-fingers is: C-X2-CL-X-E-X7-R-X2-P-X-C-X-H-X-FH-X2-C-X-D-X-W-X6-CP-X-C, where X is any amino acid. Induction of the corresponding genes is indicated as: C (chitin), F (flagellin), P (pathogens), Av (Avr9), BTH (benzothaidiazole), ACC (1-aminocyclopropane-1-carboxylic acid), CWP (cell wall protein fraction elicitor), (+) indicates mutants in these genes have been tested for altered pathogen response. doi:10.1371/journal.pone.0014426.g001 number of mature conidiophores per fungal colony compared to wild type (Figure 3A). The atl9-1, atl9-2 and atl9-3 mutations all resulted in a more pronounced susceptibility phenotype than observed in wild type plants. All plants tested were more susceptible than the resistant Kas-1 ecotype and complementation of the atl9 mutation restored the wild-type defense phenotype (data not shown). Transformed plants containing an over-expression construct of ATL9 were significantly more resistant to G. cichoracearum and showed a 2-fold decrease in conidiophores per colony compared to wild-type plants (Figure 3A). Since previously published microarray data (NASC array) indicated that expression of ATL9 in roots was compromised in the NADPH oxidase AtrbohC mutant, we tested the defense phenotype of mutants in the NADPH oxidases AtrbohD and AtrbohF which are expressed in leaves and in the double mutant AtrbohD/ F against G. cichoracearum (Figure 3A). AtrbohD, AtrbohF and the SA- compromised sid2-1 mutant showed comparable numbers of conidiophores per colony and were significantly more susceptible than atl9, the 35S:ATL9 line, Col-0 or the resistant Kas-1 accession. The AtrbohD/F double mutant was the most susceptible to powdery mildew even surpassing that of sid2-1 suggesting an additive effect of the individual mutations (Figure 3A). Because the ROS response is essential for disease resistance we decided to determine if ROS production was compromised in atl9 mutant and in the Atrboh mutants (Arabidopsis thaliana respiratory burst oxidase homolog). Infected leaves of each mutant were double stained with diaminobenzidine (DAB) to visualize H2O2 and with trypan blue to observe the spread of the fungus 7 dpi with powdery mildew (Figure 3B). Consistent with our hypothesis, H2O2 production was much lower in atl9, compared to that of Col-0 or Kas-1 plants (Figure 3B). H2O2 localization was focused at points where the fungus was attempting to penetrate the epidermal cells (Arrows, Figure 3B). DAB staining of H2O2 was not visible in AtrbohD/F or the sid2-1 mutant (Figure 3B). Hydrogen peroxide was detected both at the point of fungal penetration as well as inside some epidermal cells in Col-0 plants (Figure 3B). The highest levels of hydrogen peroxide were detected in the resistant Kas-1 ecotype (Figure 3B) with the highest levels of H2O2 localizing to points of fungal penetration and in the epidermal cells directly adjacent to penetrated cells. High H2O2 levels in Kas-1 correlated with a strong inhibition of fungal growth and colony development (Figure 3B). In all cases there was good inverse correlation between the conidiophores per colony (Figure 3A) and the levels of H2O2 detected in leaves via DAB staining (Figure 3C). Taken together these data indicate that ROS production in epidermal cells is necessary for effective defense against G. cichoracearum and this ROS production is impaired in the atl9 mutant, AtrbohD and AtrbohF. This provides further evidence that functional ATL9, AtrbohD and AtrbohF expression are needed for effective defenses against powdery mildew. Basal expression of ATL9 and induction by chitin depends on NADPH oxidase activity The production of reactive oxygen species (ROS) is a key characteristic of the initial defense response of plants to pathogen attack and chitin treatment is known to elicit ROS production in roots although not in leaves [32]. Several NADPH oxidases in Arabidopsis have been identified and their roles in ROS generation and plant defense characterized [33,34,35]. A search of publically available microarray data (http://affymetrix. arabidopsis.info/narrays/geneswinger.pl) revealed that expression of ATL9 in roots was repressed by 50% in the AtrbohC mutant [36] PLoS ONE | www.plosone.org 3 December 2010 | Volume 5 | Issue 12 | e14426