Journal of Experimental Botany, Vol. 53, No. 367, pp. 387-389,
February 1, 2002
© 2002 Oxford University Press
Gene Note |
Expression of a bean acid phosphatase cDNA is correlated with disease resistance
Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695-7616, USA
Received 20 September 2001; Accepted 11 October 2001
Abstract
A cDNA clone, designated Hra28 (for hypersensitive reaction associated), was identified corresponding to an RNA transcript that accumulates in bean during the hypersensitive reaction. The Hra28 cDNA is 1084 nucleotides in length and is predicted to encode an acid phosphatase of 264 amino acids. Northern analysis demonstrated that the Hra28 transcript accumulated differentially in response to bacteria which induce a hypersensitive response (HR), a bacterium which causes disease, and a Hrp- mutant which does not elicit an HR or cause disease. In contrast, the Hra28 transcript did not accumulate in response to wounding. Thus, the Hra28 gene is induced by multiple stimuli and appears to be regulated in a complex manner.
Key words: Acid phosphatase, disease resistance, gene expression, hypersensitive reaction, Phaseolus vulgaris L.
Plants have evolved sophisticated mechanisms to protect against pathogen attack. Plants perceive the presence of pathogens and rapidly trigger responses that have been implicated as mechanisms of disease resistance. These defence responses include, but are not limited to, the production of phytoalexins, lytic enzymes, proteinases, pathogenesis-related proteins, active oxygen species, the hypersensitive response, and various plant cell wall components (Goodman and Novacky, 1994
; Klessig and Malamy, 1994; Hammond-Kosack and Jones, 1996). Although knowledge of the molecular and biochemical basis of plant disease resistance has increased significantly during the last decade, the understanding of resistance is at best rudimentary. Thus, the continued identification and study of novel components of defence has the potential to provide new and useful insights into understanding plant disease resistance, as well as strategies for enhancing resistance to economically important pathogens via genetic engineering.
Bean (Phaseolus vulgaris L.) is resistant to the bacterial plant pathogen Pseudomonas syringae pv. tabaci. This bacterium is unable to grow or cause disease when in contact with bean. A number of defence responses, including the hypersensitive response (HR), are rapidly activated when bean is inoculated with P. syringae pv. tabaci (Jakobek and Lindgren, 1993). The HR is a form of programmed cell death at the site of infection that is believed to limit the multiplication and spread of invading pathogens (Goodman and Novacky, 1994). The specific way in which the HR and other defence responses contribute to disease resistance in bean remains to be ascertained. In order to understand the resistance mechanisms of bean more fully, cDNAs to genes expressed during the HR have been isolated and characterized. A cDNA library was generated specific to bean (cv. Red Kidney) leaf tissue undergoing an HR after infiltration with P. syringae pv. tabaci Pt11528 (Jakobek et al., 1999). This library was screened as previously described (Jakobek et al., 1999) using a combination of differential and cold-plaque screening to identify cDNAs corresponding to transcripts expressed during an HR. During this process a cDNA clone, designated Hra28 (for hypersensitive reaction associated), was identified that is predicted to encode a novel acid phosphatase (GenBank Accession No. AY055218).
The full-length Hra28 cDNA is 1084 bp long and contains a 792 bp open reading frame, a 101 bp 5'-flanking sequence and a 191 bp 3'-flanking sequence. The predicted Hra28 protein consists of 264 amino acids and has a calculated molecular mass of 30.2 kDa. A BLAST (Altschul et al., 1990) search of databases of the National Center for Biotechnology Information revealed that the Hra28 protein shares homology with several plant acid phosphatases. Acid phosphatases are ubiquitous enzymes found in plants and other organisms that catalyse the dephosphorylation of a wide variety of substrates by hydrolysis of phosphate esters (Duff et al., 1994). These enzymes are believed to have roles in energy transfer and metabolic regulation in plant cells (Duff et al., 1994). A ClustalW multiple sequence alignment (Thompson et al., 1994) between the Hra28 protein, tomato acid phosphatase 1 (Genbank accession 445113) (Williamson and Colwell, 1991), a soybean root nodule acid phosphatase (Genbank accession CAA11075) (Penheiter et al., 1998) and a putative barley acid phosphatase that is activated by chemical inducers of systemic resistance (Genbank accession CAB71336) (Beber et al., 2000) is shown in Fig. 1. Tomato acid phosphatase 1 and the soybean root nodule acid phosphatase have been experimentally shown to be functional acid phosphatases (Paul and Williamson, 1987; Penheiter et al., 1997). However, the amino acid domains of these, and other acid phosphatases, involved with enzymatic activity and substrate specificity have not been clearly defined.
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In plants, acid phosphatases (or acid phophastase activity) have been localized to many cellular compartments including vacuoles, chloroplasts, cell walls, membranes, Golgi complex, and cytoplasm (Duff et al., 1994
; Olmos and Hellin, 1997). In silico analyses were completed to provide information about the possible cellular location of the Hra28 protein. The results from analyses using the Target P (Emanuelsson et al., 2000) and PSORT (Nakai and Horton, 1999) algorithms suggest that the Hra28 protein is targeted to a membrane or the secretory pathway. Analyses using the ChloroP (Emanuelsson et al., 1999) and MITOPROT (Claros and Vincens, 1996) algorithms support these predictions. In contrast, the Predotar algorithm (http://www.inra.fr/Internet/Produits/Predotar/) predicts the Hra28 protein to be localized to the mitochondria.
Studies were completed to determine the temporal relationship between Hra28 transcript accumulation and the development of the HR in bean in response to P. syringae pv. tabaci Pt11528. Phaseolus vulgaris cv. Red Kidney plants were grown and inoculated with Pt11528 using vacuum infiltration procedures as previously described (Jakobek and Lindgren, 1993). Total RNA was isolated from bean leaf tissue and fractionated on 1% agarose/formaldehyde gels as previously described (Jakobek et al., 1999). RNA samples were blotted to Nytran N hybridization membranes (Schleicher and Schuell, Keene, N.H.), and prehybridized and hybridized at 65 °C following manufacturer's protocols. The Hra28 insert was labelled with 
-32P-dCTP by random hexamer labelling (Feinberg and Vogelstein, 1983) and used as a hybridization probe. Filters were hybridized for 18 h and then washed following the manufacturer's protocols.
A visible HR occurs in bean 10–12 h after inoculation with P. syringae pv. tabaci Pt11528. Northern analysis demonstrated that the Hra28 mRNA was detected in bean leaf tissue 2 h after treatment, and transcript levels remained relatively high as the HR developed (Fig. 2A). Similar results were observed when bean plants were inoculated with P. syringae pv. tomato JL1065 in relation to HR timing and Hra28 transcript accumulation (data not shown). Thus, Hra28 transcript accumulation in bean correlated with the production of the HR induced by P. syringae pv. tabaci and pv. tomato.
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P. syringae pv. phaseolicola is the causal agent of halo blight of bean. This bacterium grows and multiplies in bean, and induces necrotic symptoms 36–48 h after inoculation. Previous studies have demonstrated that plant defence responses are suppressed by P. syringae pv. phaseolicola or are activated more slowly in bean in response to this bacterium when compared to P. syringae pv. tabaci (Jakobek and Lindgren, 1993
; Jakobek et al., 1993). Northern analysis demonstrated that the Hra28 transcript had accumulated in bean by 24 h after inoculation with P. syringae pv. phaseolicola NPS3121 (data not shown). This compares with 2 h after treatment with Pt11528 (Fig. 2A
). Therefore, Hra28 transcript accumulation was delayed in bean in response to P. syringae pv. phaseolicola NPS3121 when compared to P. syringae pv. tabaci Pt11528.
Previously, it was demonstrated that certain defence responses were induced in bean in response to various general stimuli that do not induce the HR or disease (Jakobek and Lindgren, 1993). For instance, transcripts for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase, and chitinase accumulated, and phytoalexins were synthesized in bean in response to P. syringae pv. tabaci Hrp- mutants; these mutants lack intact Type III secretion systems and are unable to elicit an HR or cause disease (Lindgren, 1997). Northern analysis demonstrated that Hra28 transcript accumulated within 2 h after treatment with the P. syringae pv. tabaci hrp mutant Pt11528::Hrp1 (Fig. 2B). Although the timing of Hra28 transcript accumulation was similar in plants inoculated with either bacteria, the levels of transcript detected in plants inoculated with Pt11528::Hrp1 appeared to be lower than that detected in plants inoculated with Pt11528 (Fig. 2B). Wounding has also been shown to induce PAL and CHS transcript accumulation in bean (Lawton and Lamb, 1987). Bean leaves were mechanically wounded 6–8 times on each half of the primary leaves by crushing with forceps, taking care to avoid crushing the midvein of the leaves. Northern analysis demonstrated that the Hra28 transcript was not detected in tissue wounded in this fashion (Fig. 2C).
The role of the putative Hra28 acid phosphatase during resistance remains to be ascertained. Acid phosphatases have been shown to be associated with disease resistance in other plants systems. For instance, transcript to a putative acid phosphatase accumulates in barley in response to chemical compounds which activate systemic resistance (Beber et al., 2000). Similarly, acid phosphatase activity increased in tobacco following inoculation with bacteria which induced an HR (Kenton et al., 1999). Acid phosphatases have a multiplicity of functions related to phosphate metabolism and an immense pool of potential substrates (Duff et al., 1994); therefore, the Hra28 gene product could have any one of a variety of roles related to disease resistance.
In summary, the Northern data presented here demonstrates that Hra28 transcript rapidly accumulated in response to bacteria which induce an HR, as well as a Hrp- mutant that does not elicit an HR or cause disease. In contrast, Hra28 transcript accumulated more slowly in response to a bacterium which causes disease. Interestingly, unlike certain defence transcripts such as PAL and CHS, the Hra28 transcript did not accumulate in response to wounding. Thus, Hra28 gene regulation in response to biotic and abiotic stress appears to be complex. Continued analysis of the Hra28 protein and other resistance-associated acid phosphatases should provide new insights into the mechanisms which protect plants against pathogen attack.
Acknowledgments
We thank Steve Antonius (Agri Sales Incorporated, Caldwell, Idaho) for providing bean seed and Tim Sit for assistance with computer graphics.
Notes
1 To whom correspondence should be addressed. Fax: +1 919 515 7716. E-mail: pete_lindgren{at}ncsu.edu 
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