We recently developed a simple method called \"agromonas\" which facilitates the detection of Pseudomonas syringae growth in agroinfiltrated leaves expressing genes of interest. This protocol consists of agroinfiltration followed by inoculation of Pseudomonas syringae by either infiltration or spray inoculation. Next, bacterial growth is measured a few days later by plating leaf extracts out on P. syringae-selective medium. In this protocol, we describe all the steps required for agroinfiltration, inoculation of P. syringae by both injection and spray infection and explain how to quantify in planta live bacteria.

The lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth, and that transient depletion of a host immune gene and transient expression of a type-III effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure-function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers a reliable approach for further comprehensive analysis of plant immunity.

Xanthomonas campestris pv. campestris (Xcc) causes the devastating disease Black rot in Brassicaceae. Typically Xcc enters the plant through specialized organs on the leaf margin, called hydathodes, and spreads from there through the vasculature. In order to mimic natural entry as closely as possible, we here describe a \"hydathode guttation\"-based entry assay for Xcc in Arabidopsis. This disease assay combines spray inoculation with the induction of guttation and allows reabsorption of guttation droplets by the plant. Moreover, our assay relies on a bioluminescent reporter strain of Xcc to allow direct visualization of both entry and subsequent spreading of Xcc in its host. The assay allows the routine infection from one to two hydathodes per Arabidopsis leaf. Infections are scored 14 days post inoculation, just before the infection goes systemic.

Sheath blight of rice, caused by Rhizoctonia solani, is one of the most important rice diseases worldwide; however, no rice cultivar has been found to be completely resistant to this fungus. To facilitate detailed analysis of sheath blight resistance at genetic, molecular, biochemical, and functional genomic levels, new methods were developed for effective and uniform infection and accurate evaluation of the disease. The efficiency of R. solani infection was tested on two resistant (Tetep and Jasmine 85) and two susceptible (Chucheongbyeo, Junambyeo) cultivars using three different inoculum types (agar block, liquid cultured mycelia ball, and mycelia suspension). By covering the inoculated sheaths with aluminum foil to maintain humidity, 100% infection rate was achieved in this study. Liquid cultured mycelia balls caused significantly longer lesions (5.4 cm) than other types of inoculum, including agar block (2.4 cm) and mycelia suspension (1.6 cm). An improved method for evaluating sheath blight disease was selected by comparing two methods for evaluating disease severity among three partially resistant cultivars and five susceptible cultivars inoculated with liquid cultured mycelia balls. In addition, a new formula was developed to calculate the disease susceptibility index. Lesion length and the susceptibility index generally were correlated in each leaf, but there were discrepancies between the two evaluation methods due to differences in plant architecture among the cultivars. The susceptibility index calculated using the new formula was the most accurate method for evaluating sheath blight disease across all cultivars. The effect of heading date and panicle number also was evaluated in relation to sheath blight resistance. Cultivars with late heading dates generally were more resistant to sheath blight than those with early heading dates.