Ralstonia solanacearum requires F-box-like domain-containing type III effectors to promote disease on several host plants

Abstract

The phytopathogenic bacterium Ralstonia solanacearum encodes a family of seven type III secretion system (T3SS) effectors that contain both a leucine-rich repeat and an F-box domain. This structure is reminiscent of a class of typical eukaryotic proteins called F-box proteins. The latter, together with Skp1 and Cullin1 subunits, constitute the SCF-type E3 ubiquitin ligase complex and control specific protein ubiquitinylation. In the eukaryotic cell, depending on the nature of the polyubiquitin chain, the ubiquitin-tagged proteins either see their properties modified or are doomed for degradation by the 26S proteasome. This pathway is essential to many developmental processes in plants, ranging from hormone signaling and flower development to stress responses. Here, we show that these previously undescribed T3SS effectors are putative bacterial F-box proteins capable of interacting with a subset of the 19 different Arabidopsis Skp1-like proteins like bona fide Arabidopsis F-box proteins. A R. solanacearum strain in which all of the seven GALA effector genes have been deleted or mutated was no longer pathogenic on Arabidopsis and less virulent on tomato. Furthermore, we found that GALA7 is a host-specificity factor, required for disease on Medicago truncatula plants. Our results indicate that the GALA T3SS effectors are essential to R. solanacearum to control disease. Because the F-box domain is essential to the virulence function of GALA7, we hypothesize that these effectors act by hijacking their host SCF-type E3 ubiquitin ligases to interfere with their host ubiquitin/proteasome pathway to promote disease.

Fig. 1.

Structure of the GALA F-box proteins. (A) Structure of the seven GALA proteins. In parentheses is the length of the protein in amino acids, followed by the number of LRR. The black box represents the 48-aa-long F-box domain as defined by Pfam. Gray boxes represent 24-aa-long LRR (14). (B) Sequence alignment of GALA F-box domains, with the GALA consensus (GALA cons.). The size of the F-box domains aligned is as defined (25). SKIP cons. stands for the consensus defined on the alignment of all F-box domains from plant proteins known to interact with ASK1 and/or ASK2 (25). hSKP2 is the human SKP2 F-box protein (Q13309). Asterisks indicate hSKP2 residues that contact hSKP1 (19). Amino acid substitution groups were numbered as follows: 1, ED; 2, NQ; 3, ST; 4, KR; 5, FYW; and 6, LIVM. Residues that are conserved >80% are dark shaded, residues that are 60–80% conserved are light shaded. Shading of hSKP2 residues according to the most conserved residue in either GALA or SKIP consensus.

Fig. 2.

Interaction between R. solanacearum GALAs and Arabidopsis ASKs. (A) Y2H among BD-GALA6, BD-GALA6ΔF-box, and AD-ASK1. Serial 10× dilutions were spotted from left to right on nonselective medium (−LT) and selective medium lacking His (−LTH). (B) Western blot probed with anti-ASK1 antibody after GST pull-down of Arabidopsis total protein extract. Equal amounts of GST, GST-GALA6, and GST-GALA6ΔF-box (as estimated by PAGE/Coomassie, data not shown) were incubated with total protein extract from Arabidopsis seedlings expressing 6myc-tagged ASK2 (23). (C) Y2H interaction pattern between ASKs and GALAs. Positive interactions were observed only when the BD was fused to GALA and the AD was fused to ASK.

Fig. 3.

Virulence test of GRS447 on Arabidopsis and tomato. (A) Progression of the average wilting symptoms on Arabidopsis Col-0 plants over 11 days; filled squares, wild-type strain GMI1000; filled triangles, GRS447. IGCs are represented as a mean and standard error calculated on three data points, at days 4, 6, and 9; open squares, GMI1000; open triangles, GRS447. (B) Plant-wilting phenotype of Arabidopsis 6 days after inoculation; GMI1000-inoculated (Upper) and GRS447-inoculated (Lower) plants. (C) Progression of the average wilting symptoms on tomato (cultivar Super Marmande) over a 12-day period, symbols as in A. (D) Tomato wilting at day 5 after inoculation; GMI1000-inoculated (Upper) and GRS447-inoculated (Lower) plants.

Fig. 4.

Virulence of GRS138 on M. truncatula line A17. Plants were inoculated at 10⁸ cfu/ml. The progression of wilting symptoms was assessed by average disease-index score. The wild-type strain GMI1000 (filled squares) induces wilting 6 days after inoculation. Strain GRS138 (filled diamonds), which carries a disruption of the GALA7-encoding gene (14), is severely impaired in its virulence on M. truncatula. GRS138 complemented with the GALA7 gene (pNP221, filled circles) has a restored virulence, whereas the inoculation with GRS138 expressing a GALA7 gene with its F-box deleted (pNP222, filled triangles) did not produce any wilting on M. truncatula. Open symbols represent the IGC (mean and standard error bars) analyzed for GMI1000 (squares) and GRS138(pNP221) (circles) at day 6 [no R. solanacearum was detected for GRS138(pNP221)] and days 10 and 15. No R. solanacearum could be detected in GRS138 or GRS138(pNP222) at these same time points.