From prediction to function: Current practices and challenges towards the functional characterization of type III effectors

Abstract

The type III secretion system (T3SS) is a well-studied pathogenicity determinant of many bacteria through which effectors (T3Es) are translocated into the host cell, where they exercise a wide range of functions to deceive the host cell's immunity and to establish a niche. Here we look at the different approaches that are used to functionally characterize a T3E. Such approaches include host localization studies, virulence screenings, biochemical activity assays, and large-scale omics, such as transcriptomics, interactomics, and metabolomics, among others. By means of the phytopathogenic Ralstonia solanacearum species complex (RSSC) as a case study, the current advances of these methods will be explored, alongside the progress made in understanding effector biology. Data obtained by such complementary methods provide crucial information to comprehend the entire function of the effectome and will eventually lead to a better understanding of the phytopathogen, opening opportunities to tackle it.

Keywords: Ralstonia solanacearum; functional characterization; immunity; pathogenicity; prediction; type III effector.

Figure 1

Retapamulin-assisted ribosome profiling (Ribo-RET) profile of S. typhimurium T3E SseL pointing to translation initiation at a newly identified upstream TIS. Bacterial ribosomes are specifically arrested at translation start sites by the antibiotic retapamulin. Here, translation initiation at a newly identified upstream TIS resulted in translation of an N-terminal extended SseL proteoform (Chromosome: 2392442–2393461). The annotated and newly discovered TISs are marked with a blue and orange arrowhead, respectively. Data from Fijalkowski et al. (2022).

Figure 2

CyaA’ translocation assay to discover T3Es. By means of transformation, the N-terminal part of the calmodulin-dependent adenylate cyclase gene (cyaA’) is randomly inserted into the genome of the bacterium. If genomic insertion disturbs effector translocation to the plant cell via the T3SS, no change in cyclic AMP (cAMP) occurs (left). However, if random insertion allows translocation to the plant cell, CyaA’ converts ATP to cAMP (right). The genome of bacteria that induced plant cAMP levels can then be queried for the presence of an effector by use of primers binding to the transposon elements flanking the cyaA’ gene (black).

Figure 3

Different experimental methods to characterize a T3E. Various methods can be applied in a complementary fashion to elucidate T3E functioning. Commonly used experimental methods are listed, but this list is not exhaustive.

Figure 4

The route for the functional characterization of RipP2. Different laboratories have contributed to the elucidation of the putative function of the T3E RipP2, formally known as PopP2, by using the different methods discussed in this review. Starting from its homology with other known acetyltransferases, the biochemical activity of the T3E (acetylation of WRKY transcription factors, including RRS1) was found as well as the catalytic residues responsible for this function. Eventually, the apo crystal structure of RipP2 or RipP2 in complex with IP₆, acetyl-CoA, and the WRKY domain from RRS1-R was determined. This figure provides an overview of the results found by Deslandes et al. (2003), Tasset et al. (2010), Le Roux et al. (2015), Xiou et al. (2015), Zhang et al. (2017), Xia et al. (2021), and Huh (2022).