Overexpression of CsSAMT in Citrus sinensis Induces Defense Response and Increases Resistance to Xanthomonas citri subsp. citri.

Citrus canker is a destructive disease caused by Xanthomonas citri subsp. citri, which affects all commercial sweet orange (Citrus sinensis [L.] Osbeck) cultivars. Salicylic acid (SA) and systemic-acquired resistance (SAR) have been demonstrated to have a crucial role in mediating plant defense responses against this phytopathogen. To induce SAR, SA is converted to methyl salicylate (MeSA) by an SA-dependent methyltransferase (SAMT) and translocated systemically to prime noninfected distal tissues. Here, we generated sweet orange transgenic plants (based on cvs. Hamlin and Valencia) overexpressing the SAMT gene from Citrus (CsSAMT) and evaluated their resistance to citrus canker. We obtained four independent transgenic lines and confirmed their significantly higher MeSA volatilization compared to wild-type controls. Plants overexpressing CsSAMT showed reduced symptoms of citrus canker and bacterial populations in all transgenic lines without compromising plant development. One representative transgenic line (V44SAMT) was used to evaluate resistance response in primary and secondary sites. Without inoculation, V44SAMT modulated CsSAMT, CsNPR1, CsNPR3, and CsWRKY22 expression, indicating that this plant is in a primed defense status. The results demonstrate that MeSA signaling prompts the plant to respond more efficiently to pathogen attacks and induces immune responses in transgenic plants at both primary and secondary infection sites.

Overexpression of mqsR in Xylella fastidiosa Leads to a Priming Effect of Cells to Copper Stress Tolerance.

Copper-based compounds are widely used in agriculture as a chemical strategy to limit the spread of multiple plant diseases; however, the continuous use of this heavy metal has caused environmental damage as well as the development of copper-resistant strains. Thus, it is important to understand how the bacterial phytopathogens evolve to manage with this metal in the field. The MqsRA Toxin-Antitoxin system has been recently described for its function in biofilm formation and copper tolerance in Xylella fastidiosa, a plant-pathogen bacterium responsible for economic damage in several crops worldwide. Here we identified differentially regulated genes by X. fastidiosa MqsRA by assessing changes in global gene expression with and without copper. Results show that mqsR overexpression led to changes in the pattern of cell aggregation, culminating in a global phenotypic heterogeneity, indicative of persister cell formation. This phenotype was also observed in wild-type cells but only in the presence of copper. This suggests that MqsR regulates genes that alter cell behavior in order to prime them to respond to copper stress, which is supported by RNA-Seq analysis. To increase cellular tolerance, proteolysis and efflux pumps and regulator related to multidrug resistance are induced in the presence of copper, in an MqsR-independent response. In this study we show a network of genes modulated by MqsR that is associated with induction of persistence in X. fastidiosa. Persistence in plant-pathogenic bacteria is an important genetic tolerance mechanism still neglected for management of phytopathogens in agriculture, for which this work expands the current knowledge and opens new perspectives for studies aiming for a more efficient control in the field.

Bacillus thuringiensis RZ2MS9, a tropical plant growth-promoting rhizobacterium, colonizes maize endophytically and alters the plant's production of volatile organic compounds during co-inoculation with Azospirillum brasilense Ab-V5.

The beneficial features of Bacillus thuringiensis (Bt) are not limited to its role as an insecticide; it is also able to promote plant growth interacting with plants and other plant growth-promoting rhizobacterium (PGPR). The PGPR Bt strain RZ2MS9 is a multi-trait maize growth promoter. We obtained a stable mutant of RZ2MS9 labelled with green fluorescent protein (RZ2MS9-GFP). We demonstrated that the Bt RZ2MS9-GFP successfully colonizes maize's roots and leaves endophytically. We evaluated whether RZ2MS9 has an additive effect on plant growth promotion when co-inoculated with Azospirillum brasilense Ab-V5. The two strains combined enhanced maize's roots and shoots dry weight around 50% and 80%, respectively, when compared to the non-inoculated control. However, non-differences were observed comparing RZ2MS9 alone and when co-inoculated with Ab-V5, In addition, we used co-inoculation experiments in glass chambers to analyse the plant's volatile organic compounds (VOCs) production during the maize-RZ2MS9 and maize-RZ2MS9-Ab-V5 interaction. We found that the single and co-inoculation altered maize's VOCs emission profile, with an increase in the production of indoles in the co-inoculation. Collectively, these results increase our knowledge about the interaction between the Bt and maize, and provide a new possibility of combined application with the commercial inoculant A. brasilense Ab-V5.

Leaping into the Unknown World of Sporisorium scitamineum Candidate Effectors.

Sporisorium scitamineum is a biotrophic fungus causing sugarcane smut disease. In this study, we set up a pipeline and used genomic and dual transcriptomic data previously obtained by our group to identify candidate effectors of S. scitamineum and their expression profiles in infected smut-resistant and susceptible sugarcane plants. The expression profile of different genes after infection in contrasting sugarcane genotypes assessed by RT-qPCR depended on the plant genotypes and disease progression. Three candidate effector genes expressed earlier only in resistant plants, four expressed in both genotypes, and three later in susceptible plants. Ten genes were cloned and transiently expressed in N. benthamiana leaves to determine their subcellular location, while four localized in more than one compartment. Two candidates, g3890 having a nucleoplasmic and mitochondrial location and g5159 targeting the plant cell wall, were selected to obtain their possible corresponding host targets using co-immunoprecipitation (CoIP) experiments and mass spectrometry. Various potential interactors were identified, including subunits of the protein phosphatase 2A and an endochitinase. We investigated the presence of orthologs in sugarcane and using transcriptome data present their expression profiles. Orthologs of sugarcane shared around 70% similarity. Identifying a set of putative fungal effectors and their plant targets provides a valuable resource for functional characterization of the molecular events leading to smut resistance in sugarcane plants and uncovers further opportunities for investigation.

Screening of tropically derived, multi-trait plant growth- promoting rhizobacteria and evaluation of corn and soybean colonization ability.

The present study assessed the plant growth-promoting (PGP) traits and diversity of culturable rhizobacteria associated with guarana (Paullinia cupana), a typical tropical plant. Ninety-six bacteria were isolated, subjected to biochemical tests, and identified by partial or total 16S rDNA sequencing. Proteobacteria and Firmicutes were the dominant rhizospheric phyla found, and Burkholderia and Bacillus were the most abundant genera. Thirteen strains exhibited the four PGP traits evaluated, and most of them belonged to the genus Burkholderia. Two multi-trait PGP strains, RZ2MS9 (Bacillus sp.) and RZ2MS16 (Burkholderia ambifaria), expressively promoted corn and soybean growth under greenhouse conditions. Compared to the non-inoculated control, increases in corn root dry weight of 247.8 and 136.9% were obtained with RZ2MS9 and RZ2MS16 inoculation, respectively, at 60days after seeding. The dry weights of corn and soybean shoots were significantly higher than those of non-inoculated plants, showing increases of more than 47% for both strains and crops. However, soybean root dry weight did not increased after bacterial inoculation with either strain. The colonization behavior of RZ2MS16 was assessed using GFP-labeling combined with fluorescence microscopy and a cultivation-based approach for quantification. RZ2MS16:gfp was able to colonize the roots and shoots of corn and soybean, revealing an endophytic behavior.