SYSTEMIC ACQUIRED RESISTANCE

 

What is Systemic Acquired Resistance?

Where resistance to a pathogen is associated with a localised necrotic lesion, the plant will subsequently be systemically  “immunized” so that further infection will either exhibit increased resistance (Fig. 1) or  reduced disease symptoms (reviewed by Ryals et al., 1996). This “systemic acquired resistance” (SAR) is associated with the systemic expression of a subset of defence genes, e.g. the acidic forms of pathogenesis-related (PR1-5) proteins (Ward et al., 1992). Search for a signal that may be mobilised from the lesion to elicit systemic resistance has led to the identification of salicylic acid (SA) as the most likely candidate. SA is synthesised to high levels around the necrotic lesion, before being (possibly) mobilised through the phloem to accumulate, at much lower levels, systemically. The fact that exogenous application of SA could induce both PR proteins and resistance to pathogen attack  was also suggestive (Fig 2).
 

Fig. 1 Systemic acquired resistance exhibited to Tobacco Mosiac Virus (TMV)  in cv. Samsun NN

 

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Legend:  Tobacco leaves of Samsun NN which had been mock- or actually challenged with TMV were inoculated on other leaves  with TMV. Depicted are lesions at seven-days post inoculation on primary (1o, previously mock-inoculated) and secondary   (2o, previously TMV inoculated) challenged plant leaves.  
 

Fig. 2. Classical model for systemic acquired resistance based on the dispersal of salicylic acid

 

                                                          Developed originally by Malamy et al., (1990)  Metraux et  al., (1990).

The Molecular Plant Pathology (MPP) Group has made some significant contributions towards the understanding of SAR and has a series of continuing projects.
 
                1.  What is the role of SA in systemic acquired resistance?
                    2.  Is SA synthesised at the lesion and then translocated systemically to induce SAR?
                    3.  How does SA increase resistance during SAR?
                    4.  What is the role of benzoic acid in SAR?
                    5.  Can plant secondary metabolism be manipulated to confer SAR? .
 
What is the role of SA in systemic acquired resistance?

In the first of a series of manipulations of plant metabolism (Fig. 3), the MPP Group developed plants expressing salicylate hydroxylase to investigate SAR. Salicylate hydroxylase, encoded by NahG, forms part of the naphthalene degradative pathway in Pseudomonas putida. This enzyme converts SA to catechol  and plants where the NahG (or the isozyme, SH-L) gene was introduced exhibited markedly reduced SA accumulation around the lesion forming at the point of infection. These plants failed to exhibit both systemic PR protein accumulation and SAR, clearly indicating the importance of SA (Bi et al., 1995).
 

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Fig.3  Route of SA biosynthesis with introduced enzymes (boxed) to manipulate the pathway.

Is SA synthesised at the lesion and then translocated systemically to induce SAR?

The neat model for SAR shown in Fig. 2 has been questioned by the work of Vernooij et al., (1994). This group showed that in grafts of  wild type scions to 35S-NahG stocks, SAR was still exhibited in the scion following infection of the stock . This  suggested that SA synthesised around the lesion (and presumably degraded by the salicylate hydroyxlase to catechol) was irrelevant to SAR (Fig. 4).

                                          Fig. 4. The results of Vernooij et al., grafting experiments

 

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To address the question of the importance of phloem-translocated SA to SAR we have attempted to fuse the  SH-L gene a series of promoters which are active in differing types of vascular tissue.  The project began by using testing the expression patterns conferred by each promoter to fused GUS reporters (Fig. 5) in tobacco.
 

Fig. 5. Variable developmental promoter-GUS expression of the vascular tissues of petioles.

In our hands, only the PR1a-promoter was active in the phloem and it was especially interesting that the 35S promoter was not  expressed in the phloem. Thus, the low level of SA which accumulated in the 35S-NahG tobacco lines used by Vernooij et al.  would be free to enter the phloem and be dispersed to confer SA (Fig. 4). A key question arising from this reinterpretation would be; how much SA is needed to generate SAR?

We have yet to resolve this question but, we have evidence from our own SAR experiments that irrespective of SA accumulation around the lesion, SH-L expression in the phloem will  reduce SAR. Our experiments were based on the using avirulent bacteria to initiate SAR (not TMV as this exhibits too great a dispersal in SH-L lines) and monitoring systemic PR protein accumulation in SH-L transgenic lines (Table 1).

Table 1:  Assessing the exhibition of SAR in SH-L expressing tobacco lines

 

Lines

SAR?

35S-SH-L 

NO

AoPR1-SH-L 

YES

PR1a-SH-L

NO

 
In line with other results SAR was abolished in 35S-SH-L,  and unsurprisingly, as the AoPR1 promoter is only transiently and locally active following infection and is not active in the phloem, the derived AoPR1-SH-L lines exhibited SAR.  However, in PR1a-SH-L where SA accumulated to high levels at the lesion (Mur et al., 1997), SAR was not exhibited.

In should be noted that this data can be intrepreted in two ways, either  the reduction of SA levels in the phloem or alternatively the systemic activitation of this promoter is sufficient to suppress the SAR. We are currently investigating which of these hypotheses are correct using transgenic lines where SH-L is only expressed in the phloem. Preliminary data suggests that the phloem is indeed a vital tissue (either as a conduit or as a site of synthesis, Smith-Becker et al., 1999) in SAR.
 
How does SA act to confer resistance?

Classically the increased resistance associated with SAR has been thought to be linked to the action of PR proteins. The function of some PR proteins is unknown, whilst others have been found to be B-glucanases and chitinases. Thus, given that B-glucans and chitin are major constituents of the fungal cell wall, such enzymes have an obvious anti-microbial role. However, it is less clear how such PR proteins could be effective against bacterial or viral pathogens. We have been exploring an additional role for SA where this acts to enhance or “potentiate” a series of defence reponses (Mur et al., 1996).
 
What is the role of benzoic acid in SAR?
 
Much data supports the hypothesis that benzoic acid (BA) is the immediate precursor of SA  (Leon et al., 1995). Following the work of Vernooij et al., (1994)  it is possible that systemic BA dispersal (BA is not a substrate for NahG) could contribute to SAR. This is currently being investigated using benzoate-4-hydroxylase expressing plants (Fig. 3) to divert BA to the defensively inactive compound, 4-hydroxbenozic acid.
 
Can plant secondary metabolism be manipulated to confer SAR?

A number of approaches are being explored that may lead to the "artificial" synthesis of SA in the absence of infection (Fig 3). One aim of this project is to synthesize SA in particular organs or tissue types which would enable us to assess the in planta mobility of SA. This approach may be provide greater insight that simply exogenously applyoing labelled SA or its precursors. It is possible that this technology could be applied to crop-plants to confer field resistance to infection.

References:

Bi, Y. M., Kenton, P., Mur, L., Darby, R., and Draper, J. (1995). Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression. Plant J 8, 235-45.

Leon, J., Shulaev, V., Yalpani, N., Lawton, M. A., and Raskin, I. (1995). Benzoic acid 2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid biosynthesis. Proc Natl Acad Sci U S A 92, 10413-7.

Malamy, J., Carr, J.P.., Klessig, D.F. and Raskin, I. (1990). Salicylic acid: A likely endogenous signal in the resistance response of tobacco to viral infection. Science 250, 1001-1004.

Metraux, J. P., Siger, H., Ryals, J., Ward, E., Wyss-Benz, M., Guadin, J., Raschdoorf, K., Schmid, E., Blum, W. and Inverardi, B. (1990). Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250, 1004-1006.

Mur, L. A. J., Naylor, G., Warner, S. A. J., Sugars, J. M., White, R. F., and Draper, J. (1996). Salicylic acid potentiates defence gene expression in tissue exhibiting acquired resistance to pathogen attack. Plant Journal 9, 559-571.

Ryals, J. A., Neuenschwander, U.H., Willits, M.G., Molina, A., Steiner, H-Y. and Hunt, M.D. (1996). Systemic Acquired Resistance. Plant Journal 8, 1809-1819.

Smith-Becker, J., Marois, E., Huguet, E. J., Midland, S. L., Sims, J. J., and Keen, N. T. (1998). Accumulation of salicylic acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems. Plant Physiol 116, 231-8.

Vernooij, B., Friedrich, L., Morse, A., Reist, R., Kolditzjawhar, R., Ward, E., Uknes, S., Kessmann, H., and Ryals, J. (1994). Salicylic-Acid Is Not the Translocated Signal Responsible For Inducing Systemic Acquired-Resistance But Is Required in Signal Transduction. Plant Cell 6, 959-965.

Ward, E., Uknes, S.J., Williams, S.C., Dincher, S.S., Wiederhold, D.L., Alexander, D.C., Ahl-Goy, p., Metraux, J-P and Ryals J.A. (1991). Coordinate gene actvity in response to agents that induce systemic acquired resistance. Plant Cell 3, 1085-1094.
 
We would like to acknowledge the invaluable contribution of Yong-Mei Bi, Simon Warner and Simon Firek, former members of the group, to this project.