Relationship between the triggering of the Stringent Response and the expression of the Type 3 Secretion System in Bradyrhizobium diazoefficiens

COLLA, DELFINA; CASTELLANI, LUCAS G.; TORRES TEJERIZO, G.; PEREZ GIMENEZ, JULIETA

Mendoza

Congreso; Congreso SAIB 2022; 2022

Bradyrhizobium diazoefficiens USDA 110 is a Gram-negative soil bacterium which fixes nitrogen insymbiosis with soybean plants (Glycine max ) in so-called root nodules. In return, the plant providesphoto-assimilated products. In B. diazoefficiens , the Type 3 Secretion System (T3SS) is inducible bygenistein, a soybean flavonoid that activates the transcriptional regulator TtsI. TtsI regulates theexpression of the T3SS, including the structural gene rhcJ, and the effector proteins involved in thesuppression of the host's defenses. Also, the T3SS induction is affected by the second messengerguanosine tetraphosphate and guanosine pentaphosphate variations (collectively referred as(p)ppGpp). In response to starvation, bacteria trigger the Stringent Response (SR) and (p)ppGpp issynthesized. A B. diazoefficiens rsh mutant, incapable of triggering the SR, establishes a defectivesymbiosis with soybean. In addition, this mutant induces far less the expression of the T3SS regulator ttsIand the rhcJ structural gene in the presence of genistein compared to the induction of the wild-type andalso, does not suppress host’s defenses.In order to find determinants linking SR and T3SS, we performed a comparative exoproteomic analysis.Extracellular proteins were obtained from wild-type (WT) and rsh mutant (MUT) strains, induced withgenistein (G) or without induction (WI). These proteins were separated and identified by Orbitrap and thespectra were analyzed with DIA-NN software. We obtained about 1000 proteins per sample (WT G, WT WI,MUT G and MUT WI), from which different comparisons allow us to obtain differentially expressed proteins.As an example, in the WT G vs MUT G comparison, we found that the T3SS’s effector protein NopE1 wasoverexpressed, indicating that T3SS is upregulated in the WT.As a connection between SR and T3SS was described, we hypothesized if the nutritional stress that triggersSR might also trigger the expression of T3SS. For this purpose, we measured the transcriptional levels ofthe ttsI and rhcJ genes of the WT and rsh mutant subjected to nutritional stress by qRT-PCR. We incubatedthe WT and the rsh mutant with genistein, without induction as a control condition and with a C and Nfree solution, condition where the (p)ppGpp is accumulated in the WT. The transcript levels of genes ttsIand rhcJ were upregulated in the WT under a nutritional stress, in comparison with the rsh mutant. Evenmore, the expression of T3SS genes under stress condition was higher than the expression in presence ofgenistein. Under nutritional starvation, the symbiotic interaction is stimulated. Our results confirmed thatin B. diazoefficiens USDA 110 there is a link between the T3SS expression and the nutrient starvationcondition, and that the expression of T3SS is triggered by the SR, but further work will help to understandthe complete pathways involved during this activation.