Using Molecular Tools To Decipher the Complex World of Plant

Article pubs.acs.org/JAFC

Using Molecular Tools To Decipher the Complex World of Plant Resistance Inducers: An Apple Case Study Thomas Dugé de Bernonville,†,‡,§,§ Brice Marolleau,†,‡,§ Johan Staub,†,‡,§,⊗ Matthieu Gaucher,†,‡,§ and Marie-Noel̈ le Brisset*,†,‡,§ †

INRA, UMR1345 IRHS, F-49071 Angers, France Université d’Angers, UMR1345 IRHS, SFR 4207 QUASAV, PRES L’UNAM, F-49045 Angers, France § Agrocampus-Ouest, UMR1345 IRHS, F-49045 Angers, France ‡

S Supporting Information *

ABSTRACT: Exogenous application of plant resistance inducers (PRIs) able to activate plant defenses is an interesting approach for new integrated pest management practices. The full integration of PRIs into agricultural practices requires methods for the fast and objective upstream screening of efficient PRIs and optimization of their application. To select active PRIs, we used a molecular tool as an alternative to methods involving plant protection assays. The expressions of 28 genes involved in complementary plant defense mechanisms were simultaneously determined by quantitative real-time PCR in PRI-treated tissues. Using a set of 10 commercial preparations and considering the pathosystem apple/Erwinia amylovora, this study shows a strong correlation between defense activation and protection efficiency in controlled conditions, thus enabling the easy identification of promising PRIs in fire blight protection. Hence this work clearly highlights the benefits of using a molecular tool to discriminate nonactive PRI preparations and provides useful molecular markers for the optimization of their use in orchard. KEYWORDS: Malus × domestica, defense, elicitor, induced resistance

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be direct or only observable during an infection process.9 In this last case, PRIs prepare the plant defense system in a so-called primed state (priming process), which leads to a stronger induction of it upon pathogen recognition.10 Plant defense elicitation has the advantage of using environmentally sound compounds to trigger defenses against a broad spectrum of pathogens and pests often in a systemic way known as systemic acquired resistance (SAR).6,11 Several commercial formulations are known to efficiently induce plant defense in controlled conditions.5−7 The best example belongs to the salicylic acid analogue family of benzothiadiazoles. The efficiency of acibenzolar-S-methyl, a benzothiadiazole derivative, has indeed been reported in many studies, with activities conferring high protection levels against viral,12 bacterial,13 and fungal pathogens.14 However, practical application in field assays demonstrated irregular efficiencies influenced by plant genotypes and environmental conditions.5−7,15 As a consequence, further analyses are mandatory to better understand the mechanisms underlying PRI mode of action to correctly integrate them, when possible, into agricultural practices in a way that maximizes their efficiency while taking into account the above-described limitations. This optimization requires nonexpensive and fast protocols able to screen candidate compounds under given physiological conditions and on a given model plant. Current screening procedures are mainly based on protection assays that may be

INTRODUCTION Plants respond to pests and pathogens through a complete set of defense mechanisms, which include both preformed and induced physicochemical barriers.1 Defense induction starts with the perception of invaders with a two-level recognition system. First, non-self determinants2,3 referred to as microbe-associated molecular patterns (MAMPs) for pathogen conserved motifs and damage-associated molecular patterns (DAMPs) for plantderived molecules modified upon infection are recognized by pattern recognition receptors (PRR). Second, specific pathogenderived effector proteins that normally act as virulence factors may be detected by resistance (R) proteins, which correspond to specific receptors able to trigger a very strong and fast defense induction upon binding with a target effector or a host protein modified by the action of this effector.4 Up to now, control of plant disease in field conditions involved conventional pesticides directly targeting a given range of bioagressors and resistant cultivars bred for the presence of R genes.1 Because of the fast-evolving ability of microorganisms, those classical strategies have been overcome in several pathosystems. In addition, environmental considerations have imposed a reduction on the use of conventional pesticides. Alternatives to these approaches include the exogenous application of plant resistance inducers (PRIs), also called plant defense activators, plant resistance activators, or elicitors. Exogenous activation of plant resistance may be achieved by the application of plant non-self determinants such as chitin oligomers or plant cell wall derived oligosaccharides to simulate the presence of pathogens5−7 or with phytohormone analogues known to regulate defense activation in a complex signaling network after pathogen perception.5−8 Defense induction may © 2014 American Chemical Society

Received: Revised: Accepted: Published: 11403

September 4, 2014 November 3, 2014 November 5, 2014 November 5, 2014 dx.doi.org/10.1021/jf504221x | J. Agric. Food Chem. 2014, 62, 11403−11411

Journal of Agricultural and Food Chemistry

Article

Table 1. Characteristics of Compounds Used in the Study

*

Concentration of commercial product.

costly in terms of time and price. Protection assays in field conditions also require infrastructures and regulatory agreements to manipulate pathogen inocula or rely on natural infections that may fluctuate from year to year. In addition, the number of candidate preparations regularly increases, and recent results suggest that new PRIs will soon be identified with the design of miniaturized cultivation systems involving the model plant Arabidopsis to test many compounds in 0.6, p < 10−6 between biological repeats). Dimension 1 clearly clustered two groups of genes, and those that strongly correlated with it (r2 > 0.5, p < 10−15) (Figure 3A) corresponded to those having the first expression pattern described above (Figure 2B). This indicated that the data set could be correctly summarized by using dimension 1 as a new variable. By comparing values of samples projected on this dimension, we observed three intensities of gene induction, according to the product applied, ranging from high (compound C) to intermediate (D, G, H, I) and low (A, B, E, F, J, K) (Figure 3B). For this last group of compounds, gene expression levels did not significantly change in most cases in comparison to water treatment. The expression pattern for compound C, which contained acibenzolar-S-methyl, a well-known SAR inducer,6,29 was expected. In addition, previous studies already reported the induction of PR-protein encoding genes by acibenzolar-S-methyl in apple trees.13,30 Concerning compounds that efficiently activate apple defenses, we found a strong interactive effect between compound and day factors (MANOVA, p < 10−7). Only four compounds, C, D, G, and H, significantly activated expression of genes belonging to the first cluster 1 day after application (smaller symbols) (Figure 3B). Significant inductions at day 2 were observed for compounds C and D, as well as G, H, and I to a lesser extent (p < 0.05). A transient effect could be attributed to compounds D and H because intensities at 2 days were always stronger than at 3 days. Interestingly, laminarin (compound D) was reported to induce both salicylic acid- and jasmonic acid-related defenses in grapevine and tobacco,31,32 whereas in apple only salicylic acid-related markers were induced. This difference of results might reveal specificities in the perception/signaling systems between plant species; however, both previous studies dealt with cell suspension cultures and favored laminarin in reaching its targets. Our data also showed that compound G triggered a priming effect, because expression levels of pattern 1 genes without H2O2 application were significantly lower (Student’s t test, p < 0.05) than after H2O2 application on days 2 and 3 (Figure 3B). Our results are in agreement with a previous study on prohexadione-Ca (compound G), which reported stronger activation of defense genes in 11408

dx.doi.org/10.1021/jf504221x | J. Agric. Food Chem. 2014, 62, 11403−11411


Article

prohexadione-Ca-treated apple trees challenged with Venturia inaequalis, the causative agent of apple scab.33 Interestingly, this study also indicated that defense genes primed with prohexadione-Ca were related to salicylic acid signaling, as observed here. Another study showed that this plant growth regulator altered flavonoid metabolism by activation of the expression of genes encoding chalcone synthase (CHS) and flavanone 3hydroxylase (FHT).34,35 However, in our conditions, this compound only slightly induced the expression of the flavonoid related genes CHS, DFR (dihydroflavonol reductase), and ANS (anthocyanidin synthase), in comparison to genes encoding PRproteins. It was quite surprising that PRIs, which triggered defense activation in our study, did not display specificities in the set of induced genes, as has been previously shown for other plant species. For example, acibenzolar-S-methyl, the active molecule of product C, and chitosan triggered specific responses in strawberry, in particular concerning the flavonoid biosynthetic pathway.35 In the light of the present results and data found in the literature, it seems that the same PRI is able to trigger specific responses in distinct hosts. Although differences in treatment application (time, concentration, method of spraying, plant material) could be the source of such discrepancies, this specificity could also reflect specific abilities of plant species to respond to elicitors. Correlation between Intensity of Gene Induction and Protection Efficiency. We next investigated to what extent the protective effect was correlated with defense induction levels (estimated with values of projected individuals on dimension 1) for the apple/E. amylovora pathosystem. To establish such a correlation, we plotted at each analysis date the protection data obtained for compounds applied 4 days before inoculation (data resulting from Figure 1) vs PCA abscissa (Figure 3B). On the whole, positive correlations between protection efficiency and defense induction were obtained at the three sampling dates, although not always significant (p values of 0.03, 0.07, and 0.05 for days 1, 2, and 3, respectively) (Figure 4). However, correlations were stronger and more significant with samples corresponding to H2O2-treated leaves (r2 = 0.55, p = 0.01; and r2 = 0.687, p = 0.003 at days 2 and 3, respectively). This increase could be explained by the priming effect of compound G, which therefore contributed more to the model when defense induction was revealed by spraying H2O2. Our results show that the higher the coordinates were on dimension 1, the higher the protective effect, with the exception of compound D. Using coordinates on dimension 2 (which represented only 14% of the initial variance) in a similar way gave only low correlation coefficients (Supporting Information Figure s2). This reinforces that defense genes represented by this dimension are not related to active defense against E. amylovora. Taken together, our results were able to discriminate compounds with no effects by testing a relatively low number of genes. It is noteworthy that the most efficient products (C, G, and H) displaying protective effects together with defense elicitation are synthetic inputs dealing with phytohormone signaling pathway. Although the effects of product C (acibenzolar-S-methyl) and G (prohexadione-Ca) were expected,13,36 the activity of product H gave interesting prospects. This preparation is an auxin analogue (naphthaleneacetic acid) used as a thinning agent in apple orchards. Its effect contrasted with the antagonistic interactions between the salicylic acid and auxin signaling pathways usually reported.37 Indeed, those three compounds, C, G, and H, triggered similar responses in our case. The fact that compound

H had a high protection efficiency together with high induction activity suggests that new disease managements could take into account the property of particular growth regulators to induce plant defenses. Activities of compounds D and H displayed contrasting profiles. Whereas both triggered moderate and transient activation of defenses in a similar way, their protection efficacies strongly differed. Indeed, compound H was able to confer a significant protection against E. amylovora, whereas compound D did not. Our hypothesis is that compound H may either induce the expression of defenses not assayed here or alter plant development in a way that reduces susceptibility of treated plants to E. amylovora. However, no particular macroscopic phenotype was seen on our apple seedlings during the time of experiment. Synergism between auxin and jasmonate signaling pathways has been reported several times.38 Because methyl jasmonate treatments were shown to protect apple seedlings against E. amylovora,39 it is possible that a set of defense commonly regulated by auxin and jasmonates is involved in the responses of apple to this bacterial pathogen. As far as compound D is concerned, it could be interesting to try to correlate the transient defense induction with a protective effect 2 days after spraying. However, and from a practical point of view, the lack of protective effectiveness observed 4 days after treatment would suggest a difficult integration in orchard disease management with the requirement of a high treatment frequency. It is interesting to point out that product A does not protect against E. amylovora or stimulate defense responses in our experimental conditions. This product belongs to the phosphite family and is registered for the control of various diseases including fire blight on apple and pear. Its mode of action is known to be complex with both direct and indirect effects.5 According to our results, it does not appear to be a PRI in apple. However, for this compound as well as for other inactive compounds in our experimental conditions, we cannot exclude dose rate effects, which were not tested here and which have been reported in several works.32,40,41 Another interesting finding to further improve the use of PRIs is that strong marker genes among the 28 assayed here were identified. In particular, PR-protein encoding genes and several secondary metabolism genes strongly responded to PRI treatments. It is known that field-grown plants often exhibit high expression levels of PR-proteins.5 However, an appropriate combination of those markers might help to uncover how plants respond to PRIs and, for example, to determine periods or organs of maximum responsiveness. Taken together, our results show that compounds failing to activate specific defense genes (and with no direct antimicrobial activity) are most unlikely to confer protection against E. amylovora. The molecular tool and the associated methodology developed in this work could therefore be proposed to agribusiness to facilitate the screening of PRIs, which are efficient against fire blight without the difficult handling of the pathogen. The strong marker genes highlighted by the screening may also be further used to test the influence of different experimental factors on PRI activity and to improve their integration into disease management programs.

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ASSOCIATED CONTENT

S Supporting Information *

Figures s1 and s2 (which completes Figure 4). This material is available free of charge via the Internet at http://pubs.acs.org. 11409



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AUTHOR INFORMATION

Corresponding Author

*(M.-N.B.) Phone: +33 2-41-22-57-13. Fax: +33 2-41-22-57-05. E-mail: [email protected]. Present Addresses

§ (T.D.B.) Université François Rabelais de Tours, EA2106 Biomolécules et Biotechnologies Végétales, F-37200 Tours, France. ⊗ (J.S.) INRA, UMR1128 Génétique et Microbiologie, F-54506 Nancy, France.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Roland Chartier, Martine Devaux, Christelle Heintz, Arnaud Indiana, and Clément Gravouil for excellent technical assistance. We are grateful to the INEM team of IRHS Angers for apple seedling production and plant maintenance.

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Using Molecular Tools To Decipher the Complex World of Plant

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