Cloning, sequence and characterization of a sunflower (Helianthus annuus. L.) pathogen-induced gene showing sequence homology with auxin-induced genes ...
Plant Molecular Biology 38: 899–903, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.
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Short communication
Cloning, sequence and characterization of a sunflower (Helianthus annuus L.) pathogen-induced gene showing sequence homology with auxin-induced genes from plants F. Mazeyrat1 , S. Mouzeyar1 , P. Nicolas1 , D. Tourvieille de Labrouhe2 and G. Ledoigt1,∗ 1 GREAT,
UA Universit´e-INRA Organisation et Variabilit´e des G´enomes V´eg´etaux, Universit´e Blaise Pascal, Campus des C´ezeaux, 24 avenue des Landais, 63177 Aubi`ere, France (∗ author for correspondence); 2 GREAT, INRA, Station d’Am´elioration des Plantes et de Pathologie V´eg´etale, 234 avenue du Br´ezet, Domaine de Crouelle, 63039 Clermont-Ferrand Cedex 2, France Received 27 October 1997; accepted in revised form 29 June 1998
Key words: Helianthus annuus, Plasmopara halstedii, plant defence, auxin-induced gene
Abstract The establishment of a plant-pathogen interaction involves changes in gene expressions in both organisms. To isolate Helianthus annuus genes whose expression is induced during processes of resistance to Plasmopara halstedii, a comparison of the expression pattern of healthy sunflowers was made with sunflowers infected with 2 races of P. halstedii, either virulent or avirulent, using differential display of mRNA. A full-length cDNA, HaAC1, representing a sunflower gene whose expression is enhanced during early stages of the incompatible interaction, was isolated. Different timing of RNA accumulation is observed between compatible and incompatible combinations. Sequence analysis and database search revealed significant homology with auxin-induced genes from plants. The expression of this gene, is also induced after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D), salicylic acid (SA) and wounding.
Downy mildew, caused by the obligate parasitic fungus Plasmopara halstedii (Farl.) Berl. et de Toni, is one of the most important diseases of sunflower, Helianthus annuus L. Resistance in sunflower is conditioned by dominant major genes, given the symbol Pl. In case of incompatible combinations (resistance), Mouzeyar et al. [16] have shown that resistance leads to a hypersensitive-like reaction with cell divisions and lignification developing around the parasite, the growth of which is inhibited. Resistance is associated with an increase in chitinase and glucanase activities [4]. This situation is not specific for downy mildew and represents a general defence response. A number of genes have been found to be up-regulated in plant cells in response to fungal attack. Many of these genes, such as pal, code for enzymes involved in synthesis of products which retard or inhibit pathogenic invasion, such as phytoalexins and lignin [9]. Others code
for proteins believed to act directly on the invading pathogen. These include certain pathogenesis-related (PR) proteins such as β-1,3-glucanase [19] and chitinase [5]. In the present work, we used a combination of DDRT-PCR [15] and 50 RACE in order to isolate a new full-length cDNA whose expression is strongly enhanced during the early stages of sunflower infection by an avirulent race of P. halstedii. Infection procedure was performed as previously described [1]. RNA was prepared from sunflower hypocotyls, harvested at various times after infection, using the method described by Bogorad et al. [3]. DDRT-PCR was carried out using total RNA extracted from control, susceptible and resistant sunflower hypocotyls harvested 24 and 48 h after infection. Reverse transcription was performed using 2.5 µM of the primer dT12 VN according to the supplier’s instructions (Gibco-BRL, Life Technolo-
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Figure 1. Comparison of the predicted amino acid sequence of HaAC1 (accession number AF030301) with the homologous proteins of A. thaliana (accession number AC002292) and tobacco (accession number P40691). Black shades represent amino acids that are identical to the protein predicted from the sunflower auxin-induced gene HaAC1 and hyphens represent gaps introduced to optimize sequence alignment.
gies, France). The resulting single-stranded cDNA was used as template for PCR amplifications using 1U Taq Polymerase (Appligene, France) for 30 cycles at 94 ◦ C for 30 s, 42 ◦ C for 1 min, 72 ◦ C for 30 s, and an additional extension period at 72 ◦ C for 5 min. The combination of primers dT12 VN and W-01 (Operon Technologies, USA) allowed the isolation of a specific cDNA band, designated HaFl1. The band was eluted and reamplified using the same combination of primers as used for the DDRT-PCR, then cloned in a T/A vector (TA cloning kit, Invitrogen, Netherlands) and sequenced. The sequence permitted the choice of a gene-specific primer 50 TTCCCTGGTGGTGAACCCAGGCCAATGC-30 for cloning the 50 end of the cDNA using Marathon cDNA amplification kit (Clontech, France). The amplified product (a single band designated HaFl2) was cloned into pGEM-T Easy Vector (Promega) and sequenced. Using 2 primers 50 -AATGGCTAGAGTTCCAAGAG-
TGAAACTG-30 and 50 -CGAGTTACATACATCATGAAACCACATG-30 , based on sequence data from HaFl1 and HaFl2 (30 and 50 ends of the cDNA respectively), the complete cDNA, named HaAC1, was amplified. The PCR programme consisted of 30 cycles at 94 ◦ C for 30 s, 60 ◦ C for 1 min and 72 ◦ C for 2.5 min with a final elongation at 72 ◦ C. The amplified products were cloned into pGEM-T Easy Vector (Promega) and sequenced. The cDNA clone, called HaAC1 (GenBank accession number AF030301), resulted in 1215 bp cDNA exhibiting an open reading frame coding for a putative 37.8 kDa polypeptide with calculated isoelectric point of 6.3. This cDNA clone revealed a high degree of amino acid sequence identity with an unpublished polypeptide described as similar to auxin-induced protein from Arabidopsis thaliana (83%, GenBank accession number AC00292), with the unpublished auxin-induced protein from tobacco (79%, Swiss-Prot accession number P406911) (Fig-
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Figure 2. Southern blot analysis of sunflower HaAC1 genes. Total DNA (10 µg) of different genotypes (PAC2, RHA266, HA89) was digested with HindIII or EcoRV and electrophoresed on a 0.8% agarose gel, blotted and hybridized with HaAC1 cDNA.
ure 1) and with the In2-2 polypeptide from maize, isolated as a safener induced gene [10]. All of these proteins are supposed to belong to aldo-keto reductase family which has been implicated in the polyols pathway [11]. Despite the overall similarity found, none of the conserved motifs typical of the aldo-keto reductase superfamily was detected in the amino acid sequence of HaAC1. Total genomic DNA, prepared from leaves of different genotypes of sunflower was digested by EcoRV and HindIII and used for Southern blotting analysis [7]. At least seven bands with differing intensities were observed after hybridization (Figure 2) indicating that HaAC1 is a member of a small multigenic family in sunflower. Changes in HaAC1 mRNA level were examined in hypocotyls of sunflower following infection with either race 1 (incompatibility) or race B (compatibility) of P. halstedii for various periods of time. The time course of transcriptional activation of this gene is shown in Figure 3. No significant accumulation of HaAC1 mRNA was seen when the plants were sprayed with water alone, indicating that the mRNA accumulation induced by the fungus was not due to the stress upon spraying. Inoculation with the avirulent race 1 resulted in the rapid accumulation (6 h) of HaAC1 mRNA in hypocotyl tissue with the maximum amounts of mRNA 24 and 48 h after infection. In the compatible combination, no increase in mRNA was observed until 48 h after inoculation, and this amount
declined to become undetectable 72 h after inoculation. Different timing of RNA accumulation is thus observed between compatible and incompatible combinations. In the compatible interaction, fungal growth is already far more advanced than in incompatible interaction indicating that in the incompatible interaction the fast accumulation of the mRNA is specifically induced by an avirulent race of P. halstedii and is not a result of non-specific stress caused by colonization of the plant by the fungus. The rapid induction of these mRNA could be the result of recognition by the plant of a race-specific elicitor produced by the fungus. In contrast, in the compatible interaction, the fungus was not inhibited and massively invaded tissue. The stress induced by fungal growth may cause a slow and delayed induction of mRNA accumulation. Differential expression of defence genes during compatible and incompatible plant-pathogen interaction is frequently observed [9, 13]. For example, accumulation of defence-related transcripts, chitinase mRNA, from pea leaves inoculated with Ascochyta pisi, was shown to be later in compatible interactions than in incompatible ones [21]. Therefore, the mechanisms of plant defence not only include different types of gene expression, but also develop specific sequences of events in time. This downy mildew-induced gene sequence has been shown to be related to auxin-induced genes. To check this hypothesis, changes in HaAC1 mRNA levels were examined in excised hypocotyls incubated in 200 ml of buffer (10 mM potassium phosphate pH 6, 2% (w/v) sucrose) with or without 2,4-D (1 µM, 10 µM or 50 µM) at 18 ± 1 ◦ C with continuous shaking. Incubation of excised hypocotyls with 1 µM, 10 µM or 50 µM of 2,4-D resulted in the accumulation of HaAC1 mRNA within 1 h as shown in Figure 4, however the induction with 1 µM of 2,4-D was weaker. These results indicate that HaAC1 gene codes for transcripts that increase considerably after auxin application. Auxin, a plant hormone, regulates various growth and developmental processes in higher plants. Expression of a number of cDNA clones have been shown to be regulated by auxins [18] and many are involved in defence-related reactions in plants. HaAC1 gene activation during infection could be a consequence of hormone production. Several microbial infections are accompanied by indole-3-acetic acid (IAA) accumulation [17] and in the resistant tomato cv. Davis, inhibition of Fusarium oxysporum growth in dual culture and conidia germination are higher when the hormonal balance is modified in favour of
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Figure 3. Northern blot hybridization of total RNA H. annuus hypocotyls infected with P. halstedii (i, incompatible interaction with race 1; c, compatible interaction with race B) and uninfected (u) at the times indicates (h). 10 µg of total RNA were separated on formaldehyde agarose gels, blotted and hybridized with HaAC1 cDNA (A). Equal loading of RNA was confirmed by hybridization with cDNA probe corresponding to the 18S ribosomal RNA (B).
auxins [20]. Benz and Spring [2] have isolated an IAA oxidase, in systemic infected sunflower (susceptible), and shown that application of exogenous auxins, such as IAA or 2,4-D, was ineffective to stimulate growth of infected seedlings. They suggest that IAA-oxidase, inactive in healthy sunflower, could be induced by the pathogen. However, phenolic compounds affect IAA oxidation [14] and these compounds were found to accumulate in resistant sunflowers [16]. These results could explain the induction of HaAC1 by auxin in the early stages of the incompatible combination. Additionally, some auxin-induced genes have been found to be induced by different stresses such as salicylic acid (SA) application or wounding [18]. SA is known to act as a signal for defence mechanisms in plant and triggers PR genes expression and resistance [12]. For these purposes, we tested whether the HaAC1 gene was induced by these stresses. Sunflower leaves were sprayed with SA (5 mM) or wounded using sandpaper. Northern analysis showed that SA induced a rapid expression of HaAC1 mRNA (1 h) with a maximum accumulation 10 hours after treatment (Figure 5) and that HaAC1 gene was also rapidly induced after wounding (Figure 6). These results suggest that HaAC1 gene is not exclusively induced by auxin but also may be inducible by other stress and contrast with the finding of De Veylder et al. [6] who reported that In2-2 (homologous with HaAC1) is not inducible by auxin or stress. As suggested by Sitbon and Perrot-Rechnman [18], auxin could influence gene expression through more than one mechanism. In addition, in the tobacco multiple stimulus response gene str246C, a region was found to be necessary for induction of promoter activ-
Figure 4. Northern blot hybridization of total RNA from H. annuus hypocotyls incubated for 1 or 3 hours in absence of 2,4-D (−2,4-D) or incubated for 1 or 3 hours with 2,4-D (+1 µM, 10 µM or 50 µM). 10 µg of total RNA were separated on formaldehyde agarose gels, blotted and hybridized with HaAC1 (A) or 18S (B) cDNA probes.
ity in response to Pseudomonas solanacearum while auxin inducibility was not controlled by this element suggesting the existence of two independent pathways for hormonal and infection inducibilities [8]. In conclusion, a new pathogenesis-related gene, also induced by 2,4-D, SA and wounding, was cloned and correlation between its expression and the resistance of sunflower to P. halstedii was found. However, the role of HaAC1 in defence mechanisms remains to be clarified. For this, we are attempting to produce a recombinant protein corresponding to this gene to test its biological activity in vitro.
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8. Figure 5. Northern blot hybridization of total RNA from H. annuus leaves sprayed with water (control) or 5 mM SA (SA) and harvested 1 h, 3 h, 10 h and 24 h after treatments (1, 3, 10, 24). 10 µg of total RNA were separated on formaldehyde agarose gels, blotted and hybridized with HaAC1 (A) or 18S (B) cDNA probes.
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12. Figure 6. Northern blot hybridization of total RNA from H. annuus leaves unwounded (0) or wounded using sandpaper and harvested 1 h, 3 h and 10 h after wounding (1, 3, 10). 10 µg of total RNA were separated on formaldehyde agarose gels, blotted and hybridized with HaAC1 (A) or 18S (B) cDNA probes.
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Acknowledgements 16.
We would like to thank INRA and CETIOM for financing the doctoral thesis of the first author and F. Vear for her critical reading of the manuscript. This work was supported by grants from the CETIOM and the MENESER (Actions Coordonnées Sciences du Vivant).
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