New insights into Plant Growth Promoting Rhizobacterium Bacillus thuringiensis RZ2MS9 biology: entomopathogenic activity and molecular interaction with Zea mays L.

• Ano de defesa: 2019
• Autor(a) principal: Longatto, Daniel Prezotto
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: miaA gene; Bioinsecticide; Bioinseticida; Effector proteins; Força de dreno; Gene miaA; Plant health; Proteínas efetoras; Saúde vegetal; Sink strength
• Link de acesso: http://www.teses.usp.br/teses/disponiveis/11/11137/tde-23032020-143407/

The use of bioinoculants into the fields can increase crop final productivity by enhancement of a myriad of host processes, including defense against pests and diseases and improved access to soil resources and nutrients. Yet, the majority of plant-benefical microrganisms relationship studies focused on bacteria involved in nodulation in Fabaceae despite of higher potential of other inoculants. Hence, this work progressed understanding of benefical relationship between tropical free-living Plant Growth Promoting Rhizobacterium (PGPR) Bacillus sp. RZ2MS9 and plants in a multidisciplinary way. In the first chapter, we provided the first description of RZ2MS9 entomopathogenic potential corroborating its classification as a Bacillus thuringiensis strain. Briefly, the detected cuboid and spherical RZ2MS9 Insecticidal Crystal Proteins (ICP) killed Diatraea saccharalis, Helicoverpa armigera, Agrotis ipsilon and Anthonomus grandis larvae similarly to commercial bioinsecticide DiPel® (Bacillus thuringiensis serovar kurstaki HD1) in rearing essays. Additionally, a region with 67% identity to the Bacillus thuringiensis Cry1B protein pathogenic against insects of orders Lepidoptera and Coleoptera plasmid was sequenced from RZ2MS9 plasmid. In the second chapter, the in silico identification and transcriptional profile of twelve candidate effector proteins coding genes from Bacillus thuringiensis RZ2MS9 was performed during interaction with maize crop host in a gnotobiotic system. RZ2MS9 candidate effector genes coded sequences harbored three MEME exclusive motifs, and were predicted to target host cytoplasm (54.5%), apoplast (27.3%), chloroplast (9.1%) and endoplasmic reticulum (9.1%) according to LocTree3. Six RZ2MS9 candidate effector genes were associated to putative genomic islands. Overall, 45% of candidate effector genes transcripts were up-expressed at 12 and/or 120 hours post-inoculation (h.p.i.), OGY05372.1 and OGY05572.1 at both times, corroborating the efficiency of pipelines and providing runners for future studies of crop host interaction. In the third chapter, maize-RZ2MS9 benefic interaction was evaluated considering transcriptional profile of maize genes and plant growth promotion indicators such as dry matter, soluble sugars and chlorophyll. RZ2MS9 modulated the expression of different genes in leaves and roots, comparing to control, favoring roots higher sink strength and growth at V2 stage under greenhouse conditions. Down-expression of ciszeatin-transglicosidase in bacterized roots suggested reduced inactivation of cytokinin zeatin, related to chlorophyll production. Up-expression of lox, pr1, and beta-glucosidase bglu60.1 in leaves of bacterized plants suggested defense activation due to host recognition of RZ2MS9. Transcripts of RZ2MS9 gene miaA, a genetic marker of microbial cytokinin production, were detected in leaves and roots of bacterized maize seedlings. Higher chlorophyll content observed in bacterized maize seedlings cultivated in greenhouse suggestted microbial interference into host hormonal balance through a mechanism to be further studied in which host release of hormone bound forms in roots and leaves, and direct production of cytokinin by the bacterium might participate. Maize genes studied with differential expression under RZ2MS9 interaction might contribute to further studies in other maize-PGPR systems.