Ubiquitin ligase-associated protein SGT1 is required for host and nonhost disease resistance in plants

Abstract

Homologues of the yeast ubiquitin ligase-associated protein SGT1 are required for disease resistance in plants mediated by nucleotide-binding site/leucine-rich repeat (NBS-LRR) proteins. Here, by silencing SGT1 in Nicotiana benthamiana, we extend these findings and demonstrate that SGT1 has an unexpectedly general role in disease resistance. It is required for resistance responses mediated by NBS-LRR and other R proteins in which pathogen-derived elicitors are recognized either inside or outside the host plant cell. A requirement also exists for SGT1 in nonhost resistance in which all known members of a host species are resistant against every characterized isolate of a pathogen. Our findings show that silencing SGT1 affects diverse types of disease resistance in plants and support the idea that R protein-mediated and nonhost resistance may involve similar mechanisms.

Fig 1.

Sequence of NbSGT1 and other plant SGT1 proteins. (A) Structural organization of SGT1 sequences (8): TPR, tetratricopeptide repeats; VR1 and VR2, variable domains; CS motif, CHORD protein and SGT1 specific; SGS, SGT1-specific motif. The domains encoded by NbSGT1.1 and NbSGT1.2 are shown. (B) Sequence alignment of SGT1 proteins. At, Arabidopsis thaliana; Hv, barley; Os, rice; Nb, N. benthamiana; Le, tomato. Black shading, identical residues; gray shading, similar residues. NbSGT1.1 and NbSGT1.2 are 97% identical (amino acid differences are highlighted in red), and the closest Arabidopsis homologues are AtSGT1a (60% identical and 69% similar) and AtSGT1b (58% identical and 68% similar). The colored overlines indicate the domains described in A.

Fig 2.

TRV:SGT infection causes silencing of NbSGT1. (A) Viral symptoms 28 days after TRV:00 or TRV:SGT inoculation. (B) NbSGT1 protein levels in upper leaves 21 days after TRV vector inoculation. Antibodies were raised in rats by using the SGS domain of AtSGT1a and are effective against SGT1 in Arabidopsis and barley (8); consequently, they are expected to be effective against all NbSGT1 proteins. Molecular sizes are indicated. Ponceau S staining of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was for confirmation of equal loading in each lane.

Fig 3.

NbSGT1 silencing compromises a wide range of R gene-specified defense responses. (A) N resistance against TMV. (Top) RNA gel blot showing accumulation of TMV:GFP RNA at 6 dpi on inoculated leaves (local) of N-transgenic TRV:00, TRV:N, or TRV:SGT plants. Genomic (gRNA) and subgenomic (sgRNA) TMV:GFP RNAs are indicated. Approximately 500-fold more TMV:GFP RNA occurred in TRV:N and TRV:SGT leaves than in TRV:00 leaves according to dilutions of total RNA from TMV:GFP-infected tissue into total RNA from noninfected tissue. (Middle) Ethidium bromide staining of rRNA to confirm equal loading in each lane. (Bottom) UV illumination showing systemic TMV:GFP at 15 dpi. (B) Rx resistance against PVX in Rx-transgenic plants. Figure layout and experimental procedures are as described for A. (C) Pto resistance against P. syringae pv. tabaci (avrPto) in Pto-transgenic plants. Leaves were infiltrated with P. syringae pv. tabaci (avrPto), and bacterial growth in inoculated leaves was monitored for 3 days. Each data point represents the mean ± SEM of three replicate samples. (D) Appearance of HRs elicited in TRV:00 and TRV:SGT plants. The HRs were caused by Rx, Pto, Cf-4, and Cf-9 expression with their corresponding Avr protein, or RPW8.1 and RPW8.2 coexpression, or expression of Inf1 or AvrRpt2. (E) Protein gel blot analysis showing GFP levels 3 days after Agrobacterium-mediated transient expression (agroinfiltration). Infiltrated cultures of Agrobacterium carried a 35S:GUS (β-glucuronidase) (lane 2) or 35S:GFP (lanes 3–8) construct in the T-DNA. Ponceau S staining of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) for confirmation of equal loading in each lane is shown at the bottom. (F) Chemical-induced cell death in nontransgenic TRV:00 or TRV:SGT plants caused by infiltration of 3% H₂O₂/20% ethanol/1 mM NaN₃/500 mM NaCl. All experiments involved three replicated samples, and were repeated three or more times, with each repeat giving similar results.

Fig 4.

NbSGT1 is required for some nonhost resistance responses. (A) P. syringae pv. maculicola. (Upper) Growth of bacteria in TRV:00 and TRV:SGT plants. Leaves were infiltrated with low inoculum levels and bacterial growth in inoculated leaves was monitored for 7 days. Each data point represents the mean ± SEM of three replicate samples. (Lower) leaves infiltrated with high inoculum (OD₆₀₀ = 0.05) 48 h after infiltration. (B) X. axonopodis pv. vesicatoria. Experimental details as described for A, except bacterial growth was monitored for 9 days and high inoculum concentration was OD₆₀₀ = 0.1. (C) X. campestris pv. campestris. Experimental details as described for A, except high inoculum concentration was OD₆₀₀ = 0.3. (D) Accumulation of CaMV at 14 dpi in upper noninoculated leaves of N. benthamiana plants that were TRV:00-, TRV:SGT-, or non-TRV-infected. CaMV sap was inoculated mechanically on lower leaves. Brassica napus Westar-10 are susceptible to CaMV and were used as controls. Upper leaves from three CaMV- and one mock-treated plant were subjected to DNA blot analysis. All experiments involved three replicated samples, and were repeated three or more times, with each repeat giving similar results.