The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway

Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation.


Co-immunoprecipitation assays
Yeast co-immunoprecipitations were performed as follows: overnight yeast cultures grown in inducing conditions were diluted to an OD 600 of 0.05 and resuspended in 500 µl of extraction buffer (50 mM Tris-HCl pH7.4, 100 mM NaCl, 1% Triton X-100) supplemented with 2 mM DTT, 1 mM PMSF and complete protease inhibitor (Roche, Basel, Switzerland). 300 µl of Zirconia glass beads (BioSpec Products, Bartlesville, USA) were added and the suspension homogenized using a FastPrep (MP Biomedicals, Santa Ana, USA) for 5 cycles of 45 seconds each. Samples were then centrifuged 10 minutes at 500 x g at 4ºC to remove cell debris and the resulting protein extract was diluted to 1.5 mg/ml. For AWR5-Cdc55 co-immunoprecipitation 1 ml of protein extract was incubated with 50 µl of magnetic GFP beads (MACS Miltenyi Biotec, Bergisch Gladbach, Germany) according to 1 . For Lst8-TAP 1 ml of protein extract was incubated with 60 µl of rabbit IgG agarose beads (Sigma-Aldrich, Buchs, Switzerland) for 2 h at 4ºC under constant rotation. The beads were sequentially washed: 1 time with extraction buffer, 3 times with extraction buffer supplemented with 500 mM NaCl and 2 times with a buffer containing 50 mM Tris pH 8 and 2 mM DTT. Bead elution was performed by adding 30 µl of SDS 5x loading buffer and incubating 5 minutes at 95ºC. The samples were then centrifuged 1 minute at 1200 x g and 30 µl of supernatant, containing the protein eluate, transferred to a new tube.
Plant co-immunoprecipitations were performed as in 1 . Briefly, leaves of 4week-old N. benthamiana leaves were infiltrated with Agrobacterium tumefaciens GV3101 expressing the anti-silencing vector p19 2 together with myc-TOR1 3 alone or in combination with Estradiol::AWR5-HA-citrine. awr5 expression was induced 24 hours post-infiltration by treating the leaves with 20 µM estradiol. Eight hours later 3 leaf disks per sample were snap-frozen in liquid nitrogen and 200 µl of ice-cold extraction buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) supplemented with 1 mM PMSF and complete protease inhibitor (Roche, Basel, Switzerland) was added.
Samples were ground on ice using a tissue grinder and centrifuged at 16000 x g, 10 minutes at 4ºC. The supernatant was diluted to 1.5 mg/ml and processed as described above for AWR5-Cdc55 co-immunoprecipitations.

Actin staining
For localization of actin, cells were grown to early log phase, shifted to the appropriate temperature for 2 hours, fiexd in 3.7% formaldehyde, and stained with rhodamine-phalloidin (Molecular Probes) as described previously 6 . The images were obtained by fluorescent microscope using a rhodamine filter.

SD-Ura+gluc SD-Ura+gal
Supplementary Figure S1. Full-length AWR5 causes growth inhibition in yeast when expressed from a high-copy-number plasmid. Yeast strains carrying the awr5 effector gene or its N-terminal (Nt-AWR5), central (Cen-AWR5) and C-terminal (Ct-AWR5) fragments or a control (GFP) gene were subjected to serial 10-fold dilutions and spotted onto solid SD-Ura+gluc (glucose -repressing medium) and SD-Ura+gal (galactose -inducing medium). Photographs were taken after 2 days of growth. Yeast wild-type cells carrying pRS425-Ptetoff-AWR5 plasmid or tor2 ts mutant cells were grown in SD (-Leu) with or without doxycycline (20 μg/ml) to early log phase, shifted to the appropriate temperature for 2 hours, fixed and stained with rhodamine-phalloidin. Bar, 5 μm