Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

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Cardiovascular disease is the leading cause of death worldwide. Nitric oxide (NO) plays a fundamental role in cardiovascular health and disruptions in normal NO physiology is associated with the progression of cardiovascular disease. A relatively new mechanism by which heme proteins can support vasodilatation during hypoxia is by converting nitrite (NO2‐) to NO. This NO2‐ reductase activity plays essential roles in a variety of physiological processes. Because there is a large range in NO2‐ affinity and NO2‐ reductase activity in heme proteins with the same active site, this research seeks to better understand how the protein environment controls the binding chemistry of NO2‐ to the heme active site. Our goal is to determine how the distal pocket environment affects the binding affinity of NO2‐ to metmyoglobin mutants. Our hypothesis is that the distal pocket environment can increase the binding affinity of NO2‐ by way of electrostatic and steric interactions. To do this, we will compare the binding affinity of a series of distal pocket mutants that affect hydrogen bonding, polarity, and the size of the distal pocket. We will correlate coordination chemistry and binding affinity in order to establish a clear picture of how the protein environment controls NO2‐ binding affinity in heme proteins. We found that polar distal residues exhibit greater binding affinity to NO2‐ than non‐polar residues due to hydrogen bonding. These results indicate that proteins with polar distal residues will exhibit greater NO2‐ reductase activity. Understanding how the protein environment influences NO2‐ binding in heme proteins helps toward understanding how these proteins generate NO physiologically, and in designing therapeutics based on the NO2‐ reductase activity of heme proteins.

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