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The most predictable mechanism of gene-for-gene resistance would be through a direct receptor–ligand interaction between R protein and Avr effector.

However, direct interactions have only been demonstrated in a few cases. Such interactions have been shown in yeast two-hybrid experiments for the flax rust resistance L5 and L6 proteins with the corresponding M. linii AvrL567 proteins. These studies showed that the TIR-NB-LRR proteins encoded by different alleles of the flax L gene interacted with the gene products of the corresponding alleles of the flax rust AvrL567 gene, and that these interactions were specified by the LRR domain (Dodds et al., 2006). Additional yeast two-hybrid interactions were demonstrated for the Arabidopsis RRS1-R protein and its cognate Avr, the R. solanacearum PopP2 protein, for the rice Pi-ta protein and its Avr from the rice blast fungus M. grisea (AvrPi-ta), and for the P50 subunit of the tobacco mosaic virus (TMV) replicase and the N gene product (Jia et al., 2000; Deslandes et al., 2003; Dodds et al., 2006; Ueda et al., 2006). The P50 interaction was observed with full-length N protein or with a construct having the TIR domain deleted and interaction between P50 and the latter construct was supported by an in vitro pull-down assay (Ueda et al., 2006). The Pi-ta interaction was narrowed down to the C-terminal LRR domain by a deletion series tested in a yeast two-hybrid assay, and direct interaction between AvrPi-ta and full-length Pi-ta was supported by probing AvrPi-ta immobilized on membranes with Pi-ta purified from bacteria (Jia et al., 2000). No direct interactions have been demonstrated either in planta or in vitro with proteins purified from plant cells, suggesting that other regions within the NB-LRR proteins or additional host factors could regulate R–Avr interactions when the proteins are expressed within the correct cellular context.

For many R genes, there have been (largely unpublished) unsuccessful attempts to demonstrate direct interactions between NB-LRR and Avr proteins through numerous approaches. Several findings have instead supported a model of indirect recognition, whereby recognition is mediated by a second host protein that physically interacts with the NB-LRR protein (Dangl and

112 M.A. Sacco and P. Moffett Jones, 2001). One model of indirect recognition, the guard hypothesis, proposes that the NB-LRR monitors the status of a host protein that is the target of the virulence activity of the Avr effector protein: the NB-LRR protein guard detects the changes effected by the activity of the cognate Avr determinant on the target protein under surveillance (guardee) (van der Biezen and Jones, 1998b). However, of the proteins identified so far as cofactors in indirect recognition (Table 5.5), none have been shown to be key targets to promote virulence in a susceptible background.

Interestingly, recognition cofactors interact with the amino-terminal domains of their cognate NB-LRR partners (Table 5.5). One of the best studied of these proteins is RIN4 from Arabidopsis, which interacts with the CC domain of the CC-NB-LRR protein RPM1 (Mackey et al., 2002; Axtell and Staskawicz, 2003). RIN4 also interacts with the two Avr proteins, AvrB and AvrRpm1, and this interaction appears to activate the RPM1 protein (Mackey et al., 2002). RIN4 also interacts with the CC-NB-LRR protein RPS2 (Axtell and Staskawicz, 2003). The Avr determinant for RPS2 is the cysteine protease AvrRpt2, which activates RPS2 upon cleavage of RIN4 (Axtell et al., 2003).

Cleavage of RIN4 also weakly activates RPM1, suggesting that both proteins respond to alterations of their recognition cofactor, RIN4, by Avr proteins (Axtell et al., 2003; Mackey et al., 2003). Possibly similar to RPS2 activation, the PBS1 kinase, which interacts with the CC domain of the Arabidopsis CC-NB-LRR protein RPS5, is also cleaved by the proteolytic activity of AvrPphB, the cognate Avr of RPS5 (Shao et al., 2003).

Another well-studied indirect interaction is that of two P. syringae effector proteins, AvrPto and AvrPtoB, with the tomato protein Prf, which makes use of the protein kinase Pto as recognition cofactor (Pedley and Martin, 2003).

Pto has historically been labelled an R protein because its polymorphism in tomato cultivars results in an apparent gene-for-gene relationship with AvrPto (Martin et al., 1993, 2003). Subsequent findings have shown that the tomato Prf protein is required for Pto-mediated resistance and shares homology with the typical R proteins of the non-TIR NB-LRR class, making Prf the actual R protein participating in this gene-for-gene interaction (Salmeron et al., 1996).

Like the above examples, Prf interacts with Pto through its amino-terminal domain (Mucyn et al., 2006). Observations noting that virulence activities of AvrPtoB and AvrPto occur independently of Pto suggest that recognition of these effectors does not strictly conform to the guard hypothesis model (Shan et al., 2000a; Abramovitch et al., 2003).

Using a biochemical approach, the Ran GTPase-activating protein (RanGAP2) from potato and Nicotiana benthamiana was shown to interact with the amino-terminal CC domain of the potato protein Rx (Sacco et al., 2007; Tameling and Baulcombe, 2007). This interaction was shown to be required for Rx-mediated resistance and possibly provides an example of a protein with a known function in other cellular processes that has been co-opted for pathogen recognition (Sacco et al., 2007; Tameling and Baulcombe, 2007). RanGAP2 also interacts with CC domains from the related proteins Rx2 and Gpa2. Since Gpa2 recognizes a different Avr determinant, this example suggests that the CC domain, through its associated protein, provides

Disease Resistance Genes: Form and Function113

Table 5.5. NB-LRR interacting proteins.

Protein Biochemical/putative activity NB-LRR

NB-LRR interacting domain

Effect of Avr on

NB-LRR interactor Reference(s) NRIP1 Thiosulfate sulfur

transferase

N TIR Cellular localization

altered

Caplan et al. (2008b)

Pto Kinase Prf CC Martin et al. (1993), Mucyn et al.

(2006)

PBS1 Kinase RPS5 CC Cleavage Swiderski and Innes (2001), Shao et

al. (2003), Ade et al. (2007)

RanGAP2 Ran GTPase activation Rx, Rx2 CC Unknown Sacco et al. (2007)

Gpa2 CC Unknown Tameling and Baulcombe (2007)

RIN4 Unknown RPM1 CC Phosphorylation Mackey et al. (2002)

RPS2 Not determined Cleavage Axtell et al. (2003)

WRKY1/2 Transcription factor Mla CC Unknown Shen et al. (2007)

CRT1 ATPase/chaperone HRT (Rx, RPS2,

SSI4)

NB Unknown Kang et al. (2008)

Hsp90 APTase/chaperone N LRR Unknown Hubert et al. (2003), Bieri et al.

(2004), Liu et al. (2004), De la Fuente van Bentem et al. (2005)

RPM1 Not determined

Mla1 LRR

Mla6 LRR

I-2 LRR

PP5 Protein phosphatase I-2 LRR Unknown De la Fuente van Bentem et al.

(2005)

RIN13 Unknown RPM1 NB-ARC Unknown Al-Daoude et al. (2005)

Sgt1 Chaperone Bs2 LRR Unknown Bieri et al. (2004)

Mla1 LRR Unknown Leister et al. (2005)

114 M.A. Sacco and P. Moffett an initial access point for Avr recognition, with the specific LRR determining which interacting Avr proteins will activate a given NB-LRR protein (Sacco et al., 2007; Rairdan et al., 2008).

Further evidence for an initial association of the Avr with the NB-LRR amino terminus through an interacting host protein cofactor may come from studies on the N protein, a TIR-NB-LRR from tobacco. The N protein was recently shown to interact through its TIR domain with a chloroplast protein (NRIP1) with sulfur transferase activity that in turn interacts with the Avr determinant, TMV P50 (Caplan et al., 2008b). Taken together with the reported interaction between the NB-LRR of N and P50 in yeast (Ueda et al., 2006), it is possible that in planta interactions with NB-LRR proteins are initially indirect, mediated by an amino-terminal binding protein that acts as a scaffold for R and Avr proteins to form a ternary complex that allows a direct R–Avr interaction through the LRR domains. Such a scenario may explain why Avr/R protein interactions can be detected when brought together in a heterologous system.

Lastly, CC domain-interacting proteins were described that associate with barley Mla1 and Mla6 and are members of the WRKY family of transcription factors (Shen et al., 2007). This is a striking observation since the RRS1-R protein has a WRKY domain fused C-terminal to its LRR domain (Deslandes et al., 2002), suggesting similar connections between transcriptional regulation by WRKY transcription factors and NB-LRR proteins. The association of Mla10 with HvWRKY2 appears to be dependent on coexpression Avr_A10, although it remains to be shown whether Avr recognition is direct or indirect in this case (Shen et al., 2007).

Outside of the NB-LRR classes, reliance on a third protein by the RLK protein Cf-2 for Avr detection has also been observed (Kruger et al., 2002).

The C. fulvum protein Avr2 interacts directly with the tomato Rcr3 protease and inhibits its activity. This physical interaction has been shown to be required for induction of Cf-2-mediated HR, although an interaction between Cf-2 and Rcr3 has not been demonstrated (Kruger et al., 2002; Rooney et al., 2005).

These observations suggest that indirect models of recognition developed for a few model pathosystems might be applicable to different classes of R proteins.

Additional NB-LRR-interacting proteins have been identified that are likely to serve different functions other than as acting as recognition cofactors. Some of these additional interacting proteins can be conceptually grouped as putative chaperones of NB-LRR proteins (Table 5.5). The protein Hsp90 has been well defined as a substrate-specific chaperone in other eukaryotic systems where it has been studied. Observations that plant Hsp90 and the proteins Rar1 and Sgt1 appear to be differentially required for the accumulation of a number of NB-LRR proteins, including Rx, RPM1, Mla1 and Mla6, and demonstrated interactions between these three proteins implicate them as R protein chaperones that may be part of ternary or higher order folding complexes (Tornero et al., 2002; Hubert et al., 2003; Lu et al., 2003; Azevedo et al., 2006). The CRT1 protein identified from Arabidopsis interacts with the NB domain of a number of NB-LRR proteins from both the TIR and the non-TIR

Disease Resistance Genes: Form and Function 115 class, and has an ATPase domain that most closely resembles that of Hsp90, suggesting a possible chaperone role (Kang et al., 2008). The protein phosphatase 5 (PP5), a co-chaperone of Hsp90, has been shown to interact with the tomato I-2, although an essential role for PP5 in I-2 function has not been established genetically (De la Fuente van Bentem et al., 2005). So far, the data accumulated for the above group of NB-LRR-interacting proteins suggests that they are likely to be differentially required chaperones whose collective roles are to facilitate the proper folding of the R proteins with which they interact, rather than to function themselves in signalling. None the less, the importance of these proteins for enabling Avr recognition and/or signalling must be acknowledged as they represent the majority of known essential factors required for the general function of R genes.

Indirect models of interactions between R proteins and Avr determinants provide an alternative evolutionary mode for adaptation of novel specificities from what would be expected in directly interacting protein systems. By detecting perturbations in an associated protein, such as in the guard model, or by detecting perturbations in intramolecular interactions that are stabilized by the amino-terminal interacting cofactor, it would be possible for R proteins to evolve to recognize activities of pathogen effectors that are not sequence specific. This relationship between R and Avr proteins, rather than recognition of specific ‘antigen’ epitopes, could afford a tolerance for more sequence diversity in Avr proteins, reducing the repertoire of pathogen ‘receptors’

required in plants compared to the extreme immune receptor diversification required in animal adaptive immunity. Moreover, the existence of two stages of recognition could allow expansion of specificity repertoires. An initiator inter-action between an Avr and a host protein cofactor anchored at the NB-LRR protein amino terminus, followed by a recognition interaction between the LRR and Avr provides two interfaces with the pathogen elicitor protein for diversification.

Signal initiation by NB-LRR proteins

Models of NB-LRR activation have emerged from detailed mutagenesis studies wherein mutants of NB-LRR protein are transiently expressed, usually in the model plant N. benthamiana, and sometimes as fragments. Expression of several NB-LRR proteins lacking their LRR domains results in an Avr-independent HR, suggesting that in the absence of pathogen elicitation, this domain plays an inhibitory role for NB-LRR signalling (Bendahmane et al., 2002). However, the observations that deletion of the LRR does not always result in Avr-independent activation and that the LRR is required for point mutation induced auto-activity supports a positive role for the LRR in activation (Bendahmane et al., 2002; Moffett et al., 2002; Hwang and Williamson, 2003). The structural similarity of NB-LRR proteins to the well-characterized animal protein Apaf-1 and mutagenesis studies of the NB and ARC domains have led to the concept of the NB-ARC module functioning as a ‘molecular switch’ for activation (Takken et al., 2006). In this model, recognition of the

116 M.A. Sacco and P. Moffett pathogen Avr induces a conformational change in the ARC, which causes an alteration of the nucleotide-binding state of the NB domain, resulting in a further conformation change that initiates signalling (Takken et al., 2006).

This model is supported by two NB mutations that alter in vitro ATPase activity of the NB domain, but result in I-2 autoactivation in planta (Tameling et al., 2006). Further studies are needed however to determine at what stage ATP is hydrolysed in the context of full-length proteins in planta.

A ‘perfect-fit’ model of NB-LRR activation has been put forward that was developed through studies of the Rx protein, and builds on the molecular switch model (Rairdan and Moffett, 2006; Rairdan et al., 2008). In this model, in the absence of Avr recognition, NB-LRR proteins are held in an inactive hair-trigger state through intramolecular interactions. A key observation from experiments with Rx was the elicitation of Avr-independent programmed cell death by a protein fragment encompassing the NB domain alone in the absence of the amino-terminal CC domain (Rairdan et al., 2008), which was previously thought to be the signalling moiety of the NB-LRR proteins (Takken et al., 2006). In this model, the ARC1 domain plays a role in recruiting the LRR to interact with the protein amino terminus. The NB-LRR protein remains in a constrained inactive state through the perfect fit of its ARC and LRR interactions. Elicitor recognition results in perturbation of the LRR that alters the interface between the LRR and ARC2; thus LRR perception of the Avr is sensed through the ARC2 domain, which acts as the switch that allows progression to an active state, with conformation changes resulting in release of the inhibitory intramolecular interactions and subsequent signalling through the NB domain (Rairdan and Moffett, 2006; Rairdan et al., 2008).

Unlike the signalling pathways that have been defined for transmission of signals initiated at the plasma membrane to the nucleus through mitogen-activated protein kinase (MAPK) signalling cascades (see Song et al., Chapter 2, this volume), the signalling pathways that lead to extreme resistance and HR remain to be defined. A number of proteins that act downstream of Avr recognition have been identified and defence activation is known to involve transcriptional reprogramming through a number of transcriptions factors (see Boyle et al., Chapter 4, and Parent et al., Chapter 6, this volume). However, since no direct link from an R protein to a signalling pathway is known as of yet, detailed discussion of these downstream players is beyond the scope of this chapter.

5.6 Localization in Function: Recognizing Avrs Where They