Background

Ralstonia solanacearum is considered one of the most destructive bacterial pathogens in plants worldwide due to its aggressiveness and wide host range. R. solanacearum invades host plants through their roots and colonizes their xylem vessels, finally spreading through the whole plant and leading to bacteria wilt disease (Xue et al., 2020). R. solanacearum is able to infect more than 250 plant species and is particularly well-adapted to infecting crops from the Solanaceae family, including tomato, potato, tobacco, eggplant, and other economically important crops (Mansfield et al., 2012). Experimental assays to assess the virulence of R. solanacearum in Solanaceae plants under laboratory conditions mostly rely on soil-drenching inoculation with a bacterial suspension followed by observation of disease symptoms (Morel et al., 2018). Other methods are based on the injection or infiltration of the bacterial suspension directly into the stems or leaves, bypassing the root penetration process and allowing a more sensitive and accurate quantitation of bacterial numbers in plant tissues (Morel et al., 2018). These methods have been extensively used to compare the virulence of different R. solanacearum strains in Solanaceae plants; however, the analysis of different plant genotypes relies on the availability of relevant plant mutants or transgenic plants, or otherwise requires their generation, which is usually time-consuming. We recently developed a method to generate tomato plants with transgenic roots suitable for subsequent soil-drenching inoculation with R. solanacearum, which allows for the faster generation of transgenic root material to study root invasion and the subsequent generation of disease symptoms (Morcillo et al., 2020). However, this method does not allow for accurate quantitation of bacterial replication in plant tissues. Nicotiana benthamiana, a model plant from the Solanaceae family, is well known for its suitability for transient gene expression mediated by Agrobacterium tumefaciens (Li, 2011). Therefore, we envisioned a straightforward procedure to manipulate gene expression in N. benthamiana tissues using A. tumefaciens followed by the subsequent infiltration of R. solanacearum and quantitation of R. solanacearum replication over time. Such a method should overcome two major issues: (i) many R. solanacearum strains, including the reference GMI1000 strain, are not virulent in N. benthamiana due to the recognition of two effector proteins (AvrAA and RipP1) by the immune system of this plant species (Poueymiro et al., 2009); and (ii) the inoculated R. solanacearum strain should be differentiated from the A. tumefaciens population previously infiltrated into the same leaf tissues. To overcome the first issue, we used the R. solanacearum Y45 strain, which was originally isolated from tobacco plants (Li et al., 2011) and lacks both AvrAA and RipP1, allowing it to replicate effectively in N. benthamiana leaf tissue (Sang et al., 2020). To circumvent the second issue, we transformed R. solanacearum Y45 with a plasmid that confers resistance to the antibiotic tetracycline to allow the selection of R. solanacearum Y45 colonies in solid medium while avoiding growth of A. tumefaciens. This simple method can be used to accurately determine R. solanacearum virulence in N. benthamiana tissues expressing heterologous genes (Sang et al., 2020; Wei et al., 2020), overexpressing endogenous genes, or undergoing gene silencing mediated by RNAi. The versatility of this method allows the study of bacterial virulence factors in plant tissues by expressing them in plant cells prior to bacterial inoculation, as well as genetic analysis of the contribution of plant genes to disease resistance or susceptibility.