Despite the important agronomic traits
Despite the important agronomic traits regulated by SUMO, most research studies on SUMOylation have been mainly limited to model plants, such as Arabidopsis and rice (Wang et al., 2011), due to the lack of molecular tools specific to other economically relevant plants. On the other hand, plants harboring mutations in main components of the SUMOylation machinery, such as Arabidopsis siz1 (Miura et al., 2010), mms21 (Huang et al., 2009, Ishida et al., 2009), or esd4 (Murtas et al., 2003), display severe growth defects that are dependent on salicylic 3-Deazaneplanocin accumulation (Miura et al., 2010, Villajuana-Bonequi et al., 2014). The development of tools alternative to null mutants are of great interest in overcoming these technical constraints.
Considering the relevance of SUMO as a major post-translational modification, it is expected that novel biological functions regulated by SUMO remain to be uncovered. Necrotrophic pathogens, such as Botrytis cinerea and Plectosphaerella cucumerina, promote host cell death to acquire nutrients for proliferation on dead and decaying tissues. Defense responses regulated by the salicylic acid-dependent pathway and associated to programmed cell death are effective against biotrophic pathogens; however, they benefit necrotrophic pathogens. Control of necrotrophic infections is achieved by a different set of defense responses activated by jasmonic acid and ethylene signaling (Glazebrook, 2005). Despite recent progress, how plants perceive and respond to necrotrophy is behind our understanding of plant responses to biotrophy (Mengiste, 2012).
Here, we have developed an innovative strategy for inhibiting SUMO conjugation in vivo as an alternative to knock-out mutants, which are lethal, in the case of E1-activating and E2-conjugating enzymes, or display strong pleiotropic phenotypes, in the case of E3 ligases. We have shown that SAE2UFDCt functions as a SUMO conjugation inhibitor both in vitro and in vivo in a dose-dependent manner, through a mechanism based on its ability to establish non-covalent interactions with the SUMO E2-conjugating enzyme. Our results showed that the SAE2UFDCt domain is sufficient for E2 recruitment in vivo, providing a novel molecular target for developing small molecule SUMO conjugation inhibitors. SAE2UFDCt expression is robust and stable through plant generations, and has allowed a novel post-transcriptional regulation of in vivo SUMO E2-conjugating enzyme levels to be uncovered. In addition, the study of these plants has facilitated the identification of a novel role of SUMO in defense responses against necrotrophic fungal pathogens. The use of SAE2UFDCt expressing lines have provided an advantage over the use of siz1 E3 ligase knock-out mutants by allowing the analysis of plant susceptibility to fungal pathogens under different degrees of SUMOylation inhibition. Our results indicate that SUMOylation is required for resistance to necrotrophic fungal attacks. During infection, free and conjugated SUMO, the E1-activating enzyme large subunit SAE2, and the E2-conjugating enzyme SCE1 diminished. In summary, we provide a novel strategy for SUMOylation inhibition that is easy to implement in any transformable plant regardless of its genetic complexity, which has been validated by uncovering a novel regulatory role of SUMO in defense responses to necrotrophic fungi. Our findings suggest that depleting host SUMO conjugation machinery could constitute a novel mechanism for fungal pathogenicity.
Discussion Taking advantage of the highly specific protein–protein interactions among cognate enzymes that mediate SUMO conjugation to substrates, we have developed a novel strategy for achieving inhibition of SUMO conjugation in vivo based on disruption of SUMO E1–E2 interactions. We have validated this strategy for uncovering a novel role of SUMO conjugation in defense responses to necrotrophic fungal pathogens.
Funding This work was supported by the European Research Council (ERC-2007-StG-205927) and the Spanish Ministry of Science (BIO2008-01495). L.C.-M., I.T., A.P., S.M., and N.R. were supported by research contracts through the CRAG. A.M. and J.S. were supported by predoctoral fellowships, Spanish Ministry of Education, Culture and Sport (FPU12/05292) and Ministry of Education and Science (BES-2005-6843), respectively, and A.L.S. was supported by Beatriu de Pinós post-doctoral grant of the Generalitat de Catalunya (2013 BP_B 00182). We also thank the Generalitat de Catalunya (Xarxa de Referència en Biotecnologia and 2009SGR 09626) for substantial support.