Table 2.
Results of selected transient expression assays with Pto, Prf and avrPto(B) in N. benthamiana.
| Prf (tomato)a | Pto (tomato)a | Effectora | Plant reactionb | References | Commentsc |
|---|---|---|---|---|---|
| Prf and Pto trigger a ligand-independent HR | |||||
| Prf35S | Pto35S | – | HR | Mucyn et al. (2006) | Overexpression of Pto and Prf under control of the 35S promoter leads to a ligand-independent HR, which occurs in the absence of AvrPto or AvrPtoB. Overexpression of Prf under control of the DEX-inducible promoter induces a Pto-independent HR, suggesting that Prf alone can activate downstream signaling. The PrfDEX-induced HR is presumably independent of Pto because it is not abolished upon silencing of the Pto homolog Pth1 from N. benthamiana with a tobacco rattle virus (TRV) silencing vector (Mucyn et al.2006). |
| Prf35S | PtoPnat | – | HR | Mucyn et al. (2006) | |
| Pto35S | – | – | Mucyn et al. (2006) | ||
| Prf35S | – | – | – | Mucyn et al. (2006) | |
| PrfDEX | – | – | HR | Mucyn et al. (2006) | |
| PrfDEX | Pto35S | – | HR | Mucyn et al. (2006) | |
| PrfDEX | TRV:Pth1 | – | HR | Mucyn et al. (2006) | |
| The Prf/Pto-triggered ligand-independent HR depends on the kinase activity of Pto | |||||
| Prf35S | PtoD164N/35S | – | – | Mucyn et al. (2006) | The ligand-independent HR induced by Prf and Pto depends on the kinase activity of Pto. The PrfDEX-induced HR is suppressed by the kinase-inactive PtoD164N derivative, but not by PtoG50S, which has residual kinase activity. |
| Prf35S | PtoG50S/35S | – | – | Mucyn et al. (2006) | |
| PrfDEX | PtoD164N/35S | – | – | Mucyn et al. (2006) | |
| PrfDEX | PtoG50S/35S | – | HR | Mucyn et al. (2006) | |
| Pto and AvrPto trigger an HR in N. benthamiana | |||||
| – | – | AvrPto35S | – | Rathjen et al. (1999) | Expression of Pto and avrPto in N. benthamiana induces the HR. Silencing of the Prf homolog from N. benthamiana abolishes the HR, suggesting that the elicitation of ETI by Pto and AvrPto depends on Prf. |
| – | Pto (transgene) | AvrPto35S | HR | Scofield et al. (1996) | |
| – | Pto35S | AvrPto35S | HR | Rathjen et al. (1999) | |
| TRV:Prf | Pto35S | AvrPto35S | – | Wu et al. (2004) | |
| Recognition of AvrPtoB depends on both Pto and Prf | |||||
| – | Pto35S (transgene) | AvrPtoB35S | – | Kim, Lin and Martin (2002); Mucyn et al. (2006) | Coexpression of Pto and avrPtoB does not trigger the HR, suggesting that the intrinisic Prf gene from N. benthamiana is not sufficient for the recognition of AvrPtoB. Coexpression of Prf, Pto and AvrPtoB triggers the HR, which appears earlier than the ligand-independent HR induced by Prf and Pto (see above). |
| Prf35S | – | AvrPtoB35S | – | Mucyn et al. (2006) | |
| Prf35S | Pto35S (transgene) | AvrPtoB35S | HR | Mucyn et al. (2006) | |
| AvrPtoB deleted in the E3 ubiquitin ligase domain triggers Prf-dependent defense reactions | |||||
| – | Pto35S | AvrPtoB35S | – | Abramovitch et al. (2003) | AvrPtoB1-387 lacks the C-terminal E3 ubiquitin ligase domain and triggers a Prf-dependent defense reaction, designated Rsb (resistance suppressed by AvrPtoB C terminus) (Abramovitch et al.2003). The C-terminal E3 ubiquitin ligase domain of AvrPtoB presumably suppresses the Rsb phenotype and also the recognition of AvrPtoB by Pto (Abramovitch et al.2003). Note that AvrPtoB1-308 is recognized by Pto but is not sufficient to elicit the Rsb phenotype (Abramovitch et al.2003). |
| – | Pto35S | AvrPtoB1-387/35S | HR | Abramovitch et al. (2003) | |
| – | – | AvrPtoB1-387/35S | HR | Abramovitch et al. (2003); Rosebrock et al. (2007) | |
| TRV:Prf | – | AvrPtoB1-387/35S | – | Rosebrock et al. (2007) | |
| – | Pto35S | AvrPtoB1-308/35S | HR | Abramovitch et al. (2003) | |
| – | – | AvrPtoB1-308/35S | – | Abramovitch et al. (2003) | |
| The kinase activity of Pto is required for ETI | |||||
| – | PtoG50S/35S | AvrPto35S | HR | Mucyn et al. (2006) | PtoG50S, which has residual kinase activity (Mucyn et al.2006), induces the HR in the presence of AvrPto as well as of Prf and AvrPtoB. It was, therefore, proposed that the kinase activity of Pto is dispensable for the activation of ETI and for the suppression of the AvrPtoB-mediated degradation of Pto (Mathieu, Schwizer and Martin 2014). This is in contrast to the finding that the kinase-inactive PtoD164N derivative does not trigger the HR in the presence of Prf and AvrPto or AvrPtoB. In contrast, PtoD164N triggers the HR in the presence of the E3 ligase mutant derivative AvrPtoBF479A, suggesting that the Pto kinase activity is required to evade AvrPtoB-mediated degradation. |
| – | PtoD164N/35S | AvrPto35S | – | Rathjen et al. (1999); Wu et al. (2004) | |
| Prf35S | PtoD164N/35S | AvrPtoDEX (transgene) | – | Mucyn et al. (2009) | |
| Prf35S | Pto35S | AvrPtoB35S | HR | Mathieu, Schwizer and Martin (2014) | |
| Prf35S | PtoG50S/35S | AvrPtoB35S | HR | Mathieu, Schwizer and Martin (2014) | |
| Prfpnat (transgene) | PtoD164N/Pro(Pto)d | AvrPtoB35S | – | Ntoukakis et al. (2009) | |
| Prfpnat (transgene) | PtoD164N/Pro(Pto) | AvrPtoBF479A/35S | HR | Ntoukakis et al. (2009) | |
| Degradation of Pto by AvrPtoB might depend on the Pto-binding site in AvrPtoB | |||||
| Prf35S | Pto35S | AvrPtoBF173A/35S | – | Mathieu, Schwizer and Martin (2014) | The N-terminal Pto-binding site is mutated in AvrPtoBF173A/35S. Based on the results of yeast two-hybrid studies, it was suggested that Pto binds adjacent to the E3 ligase domain of AvrPtoBF173A and is therefore degraded. In contrast, Pto is not degraded by AvrPtoBF173A/E3-LOF/35S, which contains an inactive E3 ubiquitin ligase domain. |
| Prf35S | Pto35S | AvrPtoBF173A/E3-LOF/35S | HR | Mathieu, Schwizer and Martin (2014) | |
| The kinase activity of Pto is dispensable for ETI if Pto has been made active by mutation | |||||
| – | PtoL205D/35S | – | HR | Wu et al. (2004); de Vries et al. (2006) | The Pto derivative PtoL205D with a mutation in the P+1 loop induces the HR in the absence of AvrPto. Similar results were observed with the kinase-inactive PtoL205D/D164N, suggesting that the kinase activity of Pto is dispensable for the HR induction by PtoL205D. The PtoL205D-triggered HR depends on the Prf gene from N. benthamiana because no HR induction was observed in Prf-silenced plants (TRV:Prf). |
| – | PtoL205D/D164N/35S | – | HR | Wu et al. (2004) | |
| TRV:Prf | PtoL205D/35S | – | – | Wu et al. (2004) | |
| Double phosphorylation of Pto is required for signaling | |||||
| PrfPnat (transgene) | Pto35S | AvrPtoBF479A/35S | HR | Ntoukakis et al. (2013) | The Pto derivative PtoS198A/T199A with mutations in both phosphorylation sites is an active kinase but does not trigger the HR in the presence of the E3 ubiquitin ligase-deficient AvrPtoBF479A. This suggests that both phosphorylation sites of Pto rather than the kinase activity per se are required for the induction of ETI. The phosphomimic PtoS198D/T199D derivative induces cell death in the presence of AvrPto or AvrPtoB. AvrPto also induces the HR in the presence of the kinase-inactive PtoS198D/T199D/D164N, suggesting that the kinase activity of Pto is dispensable after phosphorylation of S198 and T199. AvrPtoB is not recognized by the kinase-inactive PtoS198D/T199D/D164N derivative, which is presumably degraded by AvrPtoB. The L205D mutation in the P+1 loop of Pto leads to the induction of an AvrPto/B-independent cell death (see above), which is abolished in the presence of S198A/T199A mutations in Pto. This suggests that the phosphorylation of Pto at S198 and T199 is required for the ETI induction by the gain-of-function derivative PtoL205D. |
| PrfPnat (transgene) | PtoS198A/T199A/35S | AvrPtoBF479A/35S | – | Ntoukakis et al. (2013) | |
| PrfPnat (transgene) | PtoS198A/35S | AvrPto/B35S | Cell death | Ntoukakis et al. (2013) | |
| PrfPnat (transgene) | PtoT199A/35S | AvrPto/B35S | Cell death | Ntoukakis et al. (2013) | |
| PrfPnat (transgene) | PtoS198A/T199A/35S | AvrPto/B35S | – | Ntoukakis et al. (2013) | |
| PrfPnat (transgene) | PtoS198D/T199D/35S | AvrPto/B35S | Cell death | Ntoukakis et al. (2013) | |
| PrfPnat (transgene) | PtoS198D/T199D/D164N/35S | AvrPto35S | Cell death | Ntoukakis et al. (2013) | |
| PrfPnat (transgene) | PtoS198D/T199D/D164N/35S | AvrPtoB35S | – | Ntoukakis et al. (2013) | |
| PrfPnat (transgene) | PtoL205D/S198A/T199A/35S | – | – | Ntoukakis et al. (2013) | |
| Pto is transphosphorylated after effector binding | |||||
| PrfPnat (transgene) | Pto35S + PtoD164N/35S | AvrPto35S | – | Ntoukakis et al. (2013) | AvrPto binds to autophosphorylated Pto, which cannot be transphosphorylated by PtoD164N and does not trigger ETI. |
| PrfPnat (transgene) | PtoD164N/35S + PtoS198D/T199D/35S | AvrPto35S | Cell death | Ntoukakis et al. (2013) | AvrPto triggers ETI in the presence of the kinase-inactive PtoD164N upon coexpression of the phosphomimic Pto derivative PtoS198D/T199D. |
| PrfPnat (transgene) | PtoS198D/T199D/35S | – | – | Ntoukakis et al. (2013) | The phosphomimic PtoS198D/T199D derivative does not induce the ligand-independent HR, suggesting that phosphorylation of Pto is not sufficient and that the induction of Pto-dependent defense responses depends on the disruption of the P+1 loop of Pto. |
| PrfPnat (transgene) | PtoL205D/35S | – | Cell death | Ntoukakis et al. (2013) | PtoL205D induces a cell death because of the disruption of the P+1 loop. The authors speculate that PtoL205D is transphosphorylated by a Pto homolog from N. benthamiana. The presence of PtoD164N prevents transphopshorylation of PtoL205D and, therefore, suppresses the induction of ETI. |
| PrfPnat (transgene) | PtoL205D/35S + PtoD164N/35S | – | – | Ntoukakis et al. (2013) | |
| PrfPnat (transgene) | PtoL205D/35S + PtoS198D/T199D/35S | – | Cell death | Ntoukakis et al. (2013) | PtoL205D is transphosphorylated by the kinase-active derivative PtoS198D/T199D or PtoS198A/T199A and induces ETI. |
| PrfPnat (transgene) | PtoL205D/35S + PtoS198A/T199A/35S | – | Cell death | Ntoukakis et al. (2013) | |
a
The promoters, which were used for transient expression or the expression of transgenes, are indicated in all cases for which this information was provided in the publications. Pnat, native promoter of Prf; 35S, 35S promoter; DEX, DEX-inducible promoter; transgene, integration of the gene into the genome of N. benthamiana.
b
HR, hypersensitive response; -, no visible plant reactions.
c
For the better understanding of some of the results of the selected transient expression studies, conclusions provided by the authors of the indicated publications are shortly summarized. See also the text for details.