A Maize Cystatin Suppresses Host Immunity by Inhibiting Apoplastic Cysteine Proteases

Abstract

Ustilago maydis is a biotrophic pathogen causing maize (Zea mays) smut disease. Transcriptome profiling of infected maize plants indicated that a gene encoding a putative cystatin (CC9) is induced upon penetration by U. maydis wild type. By contrast, cc9 is not induced after infection with the U. maydis effector mutant Dpep1, which elicits massive plant defenses. Silencing of cc9 resulted in a strongly induced maize defense gene expression and a hypersensitive response to U. maydis wild-type infection. Consequently, fungal colonization was strongly reduced in cc9-silenced plants, while recombinant CC9 prevented salicylic acid (SA)-induced defenses. Protease activity profiling revealed a strong induction of maize Cys proteases in SA-treated leaves, which could be inhibited by addition of CC9. Transgenic maize plants overexpressing cc9-mCherry showed an apoplastic localization of CC9. The transgenic plants showed a block in Cys protease activity and SA-dependent gene expression. Moreover, activated apoplastic Cys proteases induced SA-associated defense gene expression in naïve plants, which could be suppressed by CC9. We show that apoplastic Cys proteases play a pivotal role in maize defense signaling. Moreover, we identified cystatin CC9 as a novel compatibility factor that suppresses Cys protease activity to allow biotrophic interaction of maize with the fungal pathogen U. maydis.

Maize death acids, 9-lipoxygenase–derived cyclopente(a)nones, display activity as cytotoxic phytoalexins and transcriptional mediators

“…To validate the microarray results and to compare 10-OPEA activity to other meaningful metabolites, we treated plants with either SA, 12-OPDA, JA, 10-OPEA, DA 0 -4:0, or SLB and compared the expression levels of pathogen-and insect-related genes at 3 h using qRT-PCR. Defense marker genes included ZmOPR2, the maize OPR most strongly up-regulated in response to SLB (40); pathogenesis-related 4b (ZmPR4b) and EF-hand Ca 2+ -binding protein (ZmCCD1), genes involved in the promotion of cell death and pathogen-elicited defense responses (41-43); GST 2 (ZmGST2) and hydrolase (ZmHYD), previously shown to be induced by both insect-and pathogen-related treatments (35,44); a stress-inducible oxidoreductase (ZmOXR); and maize cystatin 9 (ZmCC9), wound inducible protein 1 (ZmWIP1), and serine protease inhibitor (ZmSerPIN), that encode PIs inducible by biotic attack (27,45,46). The defense genes ZmPR4b, ZmOPR2, and ZmCCD1 were strongly induced by 10-OPEA and DA 0 -4:0 treatments with expression levels either statistically equivalent to or higher than those of 12-OPDA, JA, and SA ( Fig.…”

“…Given DA accumulation in necrotic tissue, comparatively high cytotoxicity, and weak induction of both ZmCC9 and ZmSerPIN, established inhibitors of cysteine protease-mediated cell death (27,45), we hypothesized that high concentrations of 10-OPEA may function as a positive mediator of cell death. To compare lesion-inducing activity, we examined leaves treated with 10-OPEA, DA 0 -4:0, DA 0 -4:1, iso-10-OPEA, 18:2, 18:3, and the phytohormones JA, 12-OPDA, and SA (1,47).…”

“…4A) (49, 50). As cell death processes are often mediated by cysteine proteases (27,28), we examined the capacity of 10-OPEA to induce cysteine protease activity using DCG-04, a biotinylated substrate that reacts with the catalytic cysteine residue of papainlike cysteine proteases. Immunoblot detection of DCG-04-labeled proteases from leaf tissue collected 8 h posttreatment with 10-OPEA showed stable induction of protease activity, indicated by two bands with the expected masses of 30 and 40 kDa (Fig.…”

“…As mediators, metabolites derived from the 9-LOX pathway in pepper (Capsicum annuum) and Arabidopsis positively regulate defense and cell death responses to diverse pathogens (21). Despite numerous studies linking the 9-LOX pathway to cell death processes (24)(25)(26), many oxylipin identities, activities, and links to enzyme activation remain unknown (27,28). As a positional isomer of 12-OPDA, the 9-LOX oxylipin 10-oxo-11,15-phytodienoic acid (10-OPDA) and the 18:2-derived 10-oxo-11-phytoenoic acid (10-OPEA) are structurally similar to jasmonates (29).…”

“…Exogenous application of 10-OPEA to plant tissues strongly induces defense genes, cysteine protease activity, and common cell death symptoms including lesions, ion leakage, and DNA fragmentation. Unlike JA and 12-OPDA, 10-OPEA and related DAs elicit significantly lower transcriptional up-regulation of multiple protease inhibitors (PIs) including the maize cystatin-9 (ZmCC9), a negative regulator of apoplastic mediated cell death (27). Overexpression of ZmCC9 reduces the extent of 10-OPEA-induced lesions and provides a mechanistic link to the cytotoxic action in maize.…”