Download Expression of a bean acid phosphatase cDNA is correlated with

Journal of Experimental Botany, Vol. 53, No. 367, pp. 387–389, February 2002
GENE NOTE
Expression of a bean acid phosphatase cDNA is correlated
with disease resistance
J.L. Jakobek and P.B. Lindgren1
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,
expression, hypersensitive reaction, Phaseolus vulgaris L.
gene
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
1
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 59-flanking sequence and a
191 bp 39-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.
In plants, acid phosphatases (or acid phophastase activity)
have been localized to many cellular compartments including
vacuoles, chloroplasts, cell walls, membranes, Golgi complex,
To whom correspondence should be addressed. Fax: q1 919 515 7716. E-mail: pete_lindgren@ncsu.edu
ß Society for Experimental Biology 2002
388
Jakobek and Lindgren
Fig. 1. Amino acid sequence alignment of the predicted Hra28 protein with tomato acid phosphatase 1 (TAcP; Genbank accession 445113), a soybean
root nodule acid phosphatase (SAcP; Genbank accession CAA11075) and a putative barley acid phosphatase (BAcp; Genbank accession CAB71336).
The mutilple alignment was performed using ClustalW (Thompson et al., 1994) and the alignment printed using BOXSHADE. Dashes indicate gaps
introduced by the program to maximize the alignment. Identical amino acids are highlighted in black and similar amino acids are shaded.
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:uuwww.inra.fruInternetuProduitsuPredotaru) 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% agaroseuformaldehyde 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 8C following
manufacturer’s protocols. The Hra28 insert was labelled with
a-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
Fig. 2. Hra28 transcript accumulation in bean leaf tissue in response to
Pseudomonas syringae pv. tabaci Pt11528 (Pst) (A), the Pseudomonas
syringae pv. tabaci Hrp mutant Pt11528::Hrp1 (Pst hrp Mutant) (B), or
wounding (C). Control treatments consisting of water infiltrated or Pst
inoculated tissue were included when needed. Total RNA was isolated
at various times after treatment, and hybridized to 32P-labelled Hra28.
The RNA was also hybridized to a bean cDNA, designated H1,
complementary to a constitutively expressed gene with unknown
function (Lawton and Lamb, 1987) to demonstrate equal RNA loading.
Expression of a bean acid phosphatase cDNA 389
correlated with the production of the HR induced by P. syringae
pv. tabaci and pv. tomato.
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.
Acknowledgements
We thank Steve Antonius (Agri Sales Incorporated, Caldwell, Idaho) for
providing bean seed and Tim Sit for assistance with computer graphics.
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