A prophage tail-like protein facilitates the endophytic growth of Burkholderia gladioli and mounting immunity in tomato.

A prophage tail-like protein (Bg_9562) of Burkholderia gladioli strain NGJ1 possesses broad-spectrum antifungal activity, and it is required for the bacterial ability to forage over fungi. Here, we analyzed whether heterologous overexpression of Bg_9562 or exogenous treatment with purified protein can impart disease tolerance in tomato. The physiological relevance of Bg_9562 during endophytic growth of NGJ1 was also investigated. Bg_9562 overexpressing lines demonstrate fungal and bacterial disease tolerance. They exhibit enhanced expression of defense genes and activation of mitogen-activated protein kinases. Treatment with Bg_9562 protein induces defense responses and imparts immunity in wild-type tomato. The defense-inducing ability lies within 18-51 aa region of Bg_9562 and is due to sequence homology with the bacterial flagellin epitope. Interaction studies suggest that Bg_9562 is perceived by FLAGELLIN-SENSING 2 homologs in tomato. The silencing of SlSERK3s (BAK1 homologs) prevents Bg_9562-triggered immunity. Moreover, type III secretion system-dependent translocation of Bg_9562 into host apoplast is important for elicitation of immune responses during colonization of NGJ1. Our study emphasizes that Bg_9562 is important for the endophytic growth of B. gladioli, while the plant perceives it as an indirect indicator of the presence of bacteria to mount immune responses. The findings have practical implications for controlling plant diseases.

Microliter spotting and micro-colony observation: A rapid and simple approach for counting bacterial colony forming units.

For enumerating viable bacteria, traditional dilution plating to count colony forming units (CFUs) has always been the preferred method in microbiology owing to its simplicity, albeit being laborious and time-consuming. Similar CFU counts can be obtained by quantifying growing micro-colonies in conjunction with the benefits of a microscope. Here, we employed a simple method of five to ten microliter spotting of a diluted bacterial culture multiple times on a single Petri dish followed by determining CFU by counting micro-colonies using a phase-contrast microscope. In this method, the CFU of an Escherichia coli culture can be estimated within a four-hour period after spotting. Further, within a ten-hour period after spotting, CFU in a culture of Ralstonia solanacearum, a bacterium with a generation time of around 2 h, can be estimated. The CFU number determined by micro-colonies observed for 106-fold dilutions or lower is similar to that obtained by the dilution plating method for 107-fold dilutions or lower. Micro-colony numbers observed in the early hours of growth (2 h in case of E. coli and 8 h in case of R. solanacearum) were found to remain consistent at later hours (4 h in case of E. coli and 10 h in case of R. solanacearum), where the visibility of the colonies was better due to a noticeable increase in the size of the colonies. This suggested that micro-colonies observed in the early hours indeed represent the bacterial number in the culture. Practical applications to this counting method were employed in studying the rifampicin-resistant mutation rate as well as performing a fluctuation test in E. coli. The spotting method described here to enumerate bacterial CFU results in reduction of labour, time and resources.