Implicación del sistema de secreción de tipo 3 de Sinorhizobium (Ensifer) fredii HH103 en la modulación de la respuesta de defensa de Glycine max cv. Williams y estudio de los efectores específicos secretados a través de este sistema

Abstract

Plants that interact with pathogenic bacteria in their natural environments have developed barriers to block or contain the infection. Phytopathogenic bacteria have evolved mechanisms to subvert these defenses and promote infection. Thus, some Gram-negative phytopathogenic bacteria use the type 3 secretion system (T3SS) to deliver proteins, called effectors, directly into the cytoplasm of the host cells. These effectors suppress plant defense responses to promote infection and growth of the pathogen (Galan and Collmer, 1999). The T3SS has also been found in some symbiotic rhizobial strains and the secreted effectors, collectively known as nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. Sinorhizobium (Ensifer) fredii HH103 is a broad-host range bacterium able to nodulate dozens of legumes including soybean, which is considered its natural host plant. This bacterium secretes at least eight proteins through the T3SS in response to inducer flavonoids (Rodrigues et al., 2007) and the synthesis and secretion of Nops is controlled by the T3SS transcriptional regulator TtsI whose transcription is NodD- and flavonoids-dependent (Deakin and Broughton, 2009). In this thesis we show that the inactivation of the Ensifer (Sinorhizobium) fredii HH103 T3SS negatively affect soybean nodulation very early in the symbiotic process, which is associated with a reduction of the expression of early nodulation genes. This symbiotic phenotype could be the consequence of the bacterial triggering of soybean defense responses associated to the production of salicylic acid (SA) and the impairment of the T3SS mutant to suppress these responses. Interestingly, the early induction of the transcription of GmMPK4, which negatively regulates SA accumulation and defense responses in soybean, in plants inoculated with HH103 could be associated to the differential defense responses induced by the parental and the T3SS mutant strain (Jiménez-Guerrero et al., 2015). S. fredii HH103 secretes at least eight Nops through the T3SS. Some of them cannot be considered real effectors, since they are components of the extracellular appendages of the T3SS machinery. In this thesis, we described for the first time a new Rhizobium-specific effector, which we have called NopI. This effector, like NopL and NopP, was Rhizobium-specific and could be of great importance in the symbiosis with Glycine max (soybean) cv. Williams and Vigna unguiculata. Besides, while inactivation of nopL or nopP was beneficial for symbiosis with these plants, the absence of both NopL and NopP was detrimental, suggesting that these effectors could exert complementary functions in the symbiotic process. We also confirmed that the expression of both nopL and nopI was regulated by inducer flavonoids and by the transcriptional regulators NodD1 and TtsI. In addition, translocation of NopL within soybean root cells was confirmed by the adenylate cyclase assay. Furthermore, we characterized the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in soybean nodules and nodulation assays showed that NopC exerted a positive effect on symbiosis with soybean. Finally, adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103 and not a component of the T3SS machinery. NopL and NopP effectors are phosphorylated by plant kinases, but their exact function in symbiosis is yet unknown (Deakin and Broughton, 2009). However, some results indicate that NopL could be involved in the modulation of the host MAPK signaling and in the suppression of premature senescence of nodules. NopP is also phosphorylated by plant kinases but its exact function in symbiosis is still unknown. However, inactivation of the S. fredii HH103 nopP gene causes an increase in the number of nodules formed in soybean (Deakin and Broughton, 2009). In this thesis, we studied the function of the Rhizobium-specific effectors NopL and NopP secreted by S. fredii HH103 in the symbiosis with soybean, which is considered its natural host plant. Both NopL and NopP were phosphorylated by soybean root kinases and the phosphorylation cascade was Ca2+- and calmodulin-dependent. While the signaling pathway that culminates in the phosphorylation of NopL included ser/thr and MAPKK kinases, in the case of NopP this pathway was composed of ser/thr and tyr kinases but not MAPKK kinases. Transient expression in Nicotiana benthamiana leaves of both nopL and nopP fused to YFP and further confocal imaging indicated that these effectors localized to the nucleus of the host cell and accumulate in nuclear foci, suggesting a possible role in plant gene regulation or responses to DNA stress. In this sense, the use of a yeast based array to determine functions of effectors indicated that NopP could be involved in microtubule-related processes and nuclear localization and migration. Finally, co-immunoprecipitation analyses of N. benthamiana NopL- and NopP-interacting proteins showed that NopL binds to proteins related to the plant immune response and also with calreticulin and NopP interacts with proteins related to nucleic acids (e. g. histone H4) or proteins related to plant immunity (GRAS2 transcription factors or cyclophilin 40).Premio Extraordinario de Doctorado U