Chitosan primes plant defence mechanisms against Botrytis cinerea, including expression of Avr9/Cf‐9 rapidly elicited genes

Abstract Current crop protection strategies against the fungal pathogen Botrytis cinerea rely on a combination of conventional fungicides and host genetic resistance. However, due to pathogen evolution and legislation in the use of fungicides, these strategies are not sufficient to protect plants against this pathogen. Defence elicitors can stimulate plant defence mechanisms through a phenomenon known as defence priming. Priming results in a faster and/or stronger expression of resistance upon pathogen recognition by the host. This work aims to study defence priming by a commercial formulation of the elicitor chitosan. Treatments with chitosan result in induced resistance (IR) in solanaceous and brassicaceous plants. In tomato plants, enhanced resistance has been linked with priming of callose deposition and accumulation of the plant hormone jasmonic acid (JA). Large‐scale transcriptomic analysis revealed that chitosan primes gene expression at early time‐points after infection. In addition, two novel tomato genes with a characteristic priming profile were identified, Avr9/Cf‐9 rapidly elicited protein 75 (ACRE75) and 180 (ACRE180). Transient and stable over‐expression of ACRE75, ACRE180 and their Nicotiana benthamiana homologs, revealed that they are positive regulators of plant resistance against B. cinerea. This provides valuable information in the search for strategies to protect Solanaceae plants against B. cinerea.

However, rapid pathogen evolution can result in the loss of efficacy of resistance sources and fungicides (Pappas, 1997;Williamson et al., 2007). In addition, the use of pesticides is strictly limited by European regulations due to human health and environment risk and hazard assessment changes. New alternative strategies are therefore needed. Exploiting the plant's defence system to provide protection against these threats has emerged as a potential strategy against pathogen infection and disease (Luna, 2016). defence priming, a mechanism that initiates a wide reprogramming of plant processes, considered to be an adaptive component of IR (Mauch-Mani et al., 2017). Priming has been demonstrated to be the most cost-effective mechanism of IR in terms of plant development as there is no direct relocation of plant resources from growth to defence until it is necessary (Van Hulten, Pelser, Van Loon, Pieterse, & Ton, 2006). Studies have already shown that low elicitor doses can enhance resistance to pests without interfering with crop production (Redman, Cipollini, & Schultz, 2001). Elicitor-induced defence priming has been demonstrated to last from a few days (Conrath et al., 2006) to weeks (Worrall et al., 2012) after treatment and even through subsequent generations (Ramírez-Carrasco, Martínez-Aguilar, & Alvarez-Venegas, 2017;Slaughter et al., 2012).
Priming can have multiple effects on plant defences, which vary depending on the type of plant-pathogen interaction. Defence priming enables the plant to fine-tune immunity responses through enhancement of the initial defences. This is achieved through different mechanisms that act at specific defence layers (Mauch-Mani et al., 2017). For instance, cell-wall fortification and effective production of reactive oxygen species (ROS) has been used as a marker for the expression of priming responses. Hexanoic acid (Hx) primes cellwall defences through callose deposition and redox processes in tomato cultivars against B. cinerea (Aranega-Bou et al., 2014). In Arabidopsis thaliana, BABA and benzothiadiazole (BTH)-induced priming is also based on an increase in callose deposition (Kohler, Schwindling, & Conrath, 2002;Ton et al., 2005). Priming also results in transcriptomic changes. Gene expression analysis of A. thaliana after BABA treatment was used to identify a transient accumulation of SA-dependent transcripts, including that of NPR1, which provides resistance against Pseudomonas syringae (Zimmerli, Jakab, Métraux, & Mauch-Mani, 2000). Changes in metabolite accumulation have been shown to mark priming of defence also. For instance, defence hormone profiling has shown that accumulation of JA and JA-derivatives mediates priming of mycorrhizal fungi (Pozo, López-Ráez, Azcón-Aguilar, & García-Garrido, 2015). Moreover, untargeted metabolomic analysis have identified different compounds, including kaempferol (Król, Igielski, Pollmann, & Kępczy nska, 2015), quercetin and indole 3 carboxylic acid (I3CA) (Gamir, Pastor, Kaever, Cerezo, & Flors, 2014), that drive priming responses. Several elicitors have been described to induce resistance mechanisms in tomato against B. cinerea. For instance, BABA has been demonstrated to provide long-lasting IR against B. cinerea in leaves (Luna, Beardon, Ravnskov, Scholes, & Ton, 2016) and in fruit (Wilkinson, Pastor, Paplauskas, Pétriacq, & Luna, 2018). In addition, the plant defence hormone JA has also been linked to short-term and long-term IR in tomato against B. cinerea Worrall et al., 2012). To date, however, few studies have investigated elicitor-induced defence priming in tomato against B. cinerea. One of them showed that Hxinduced priming is based on callose deposition, the expression of tomato antimicrobial genes (e.g., protease inhibitor and endochitinase genes), and the fine-tuning of redox processes (Aranega-Bou et al., 2014;Finiti et al., 2014). Therefore, evidence is building in tomato, that IR against B. cinerea can also be based on defence priming.
In this study, we investigated whether the chitin de-acetylated derivative, chitosan, triggers priming of defence in tomato against B. cinerea. Chitosan as a plant protection product is considered "generally recognized as safe" (Raafat & Sahl, 2009) that is effective in protecting strawberry, tomato and grape against B. cinerea (Muñoz & Moret, 2010;Romanazzi, Feliziani, Santini, & Landi, 2013). Different studies have shown that its effect on crop protection results from induction of defence mechanisms (Sathiyabama, Akila, & Einstein Charles, 2014) and direct antimicrobial activity (Goy, Britto, & Assis, 2009). However, treatments with chitosan require infiltration into the leaves to trigger a robust effect (Scalschi et al., 2015) making it an unsuitable method of application in large-scale experiments or studies that take into consideration first barrier defence strategies.
Here, we have addressed whether treatment with a water-soluble formulation of chitosan results in IR phenotypes and in priming of cell wall defence and defence hormone accumulation. In addition, wholescale transcriptome analysis was performed to identify candidate genes that are driving expression of priming. Our findings, together with the outlined characteristics of chitosan, make this substance a suitable candidate for extensive application as a component of Integrated Pests (and disease) Management (IPM) for the protection of crops against fungal pathogens.

| Plant material and growth conditions
Tomato cv. Money-maker seeds were used in the described experiments. Unless otherwise specified, seeds were placed into propagator trays containing Bulrush peat (Bulrush pesticide-free black peat, low nutrient and low fertilizer mix) and a top layer of vermiculite and left at 20 C until germination. Germinated seeds were transplanted to individual pots (24 pots of 55 mm wide × 60 mm long × 50 mm deep) containing Bulrush soil (pesticide-free compost mix and nutrient and fertilizer rich) in a growth cabinet for 16-8 h/day-night and 23 C/20 C cycle at~150 μE m −2 s −1 at~60% relative humidity (RH) and grown for 2 weeks until treatment. Nicotiana benthamiana seeds were cultivated in a similar manner specified for tomato for 16-8 h/day-night cycle; 26 C/22 C at~150 μE m −2 s −1 at~60% relative humidity (RH). Aubergine (Solanum melongena) cv. Black Beauty seeds were placed into propagators containing Bulrush peat and a layer of vermiculite on the top and incubated at 20 C for 1-2 weeks until germination. Seedlings were then transplanted to individual pots containing Bulrush soil and grown and cultivated as for tomato. Arabidopsis thaliana (hereafter referred to as Arabidopsis) Columbia-0 (Col-0) and transgenic lines were grown in a soil mixture of 2/3 Levington M3 soil and 1/3 sand for 8-16 h/day-night and 21 C/18 C cycle at~150 μE m −2 s −1 at~60% RH. Ten-day-old plants were transplanted to individual pots and grown for another 2 and a half weeks until treatment.

| Chemical treatment
All experiments were performed using a commercial, water-soluble chitosan formulation, known as ChitoPlant (ChiPro GmbH, Bremen, Germany) (Romanazzi et al., 2013;Younes et al., 2014). ChitoPlant, referred to as chitosan latterly, was freshly prepared in water to the specific concentrations (please see figure legends for details). Treatments were performed by foliar spraying of chitosan solution (with 0.01% Tween20) directly onto newly fully expanded leaves.

| Botrytis cinerea cultivation, infection and scoring
Botrytis cinerea R16 (Faretra & Pollastro, 1991) was used in all experiments and was kindly provided by Dr Mike Roberts (Lancaster University). Infections were performed in leaves that have been treated with chitosan 4 days before inoculations. Long-lasting experiments were performed in newly developed leaves that were not directly treated with chitosan. Cultivation of the fungus and infection of tomato-based experiments were performed as described . For N. benthamiana, 2-3 detached leaves were inoculated with 6 μL inoculum solution containing 2 × 10 4 spores/ml of B. cinerea. Infected leaves were kept at 100% RH by sealing the trays and placed in the dark before disease assessment. Arabidopsis infections were performed as previously described (La Camera et al., 2011) with a few modifications. Leaves were inoculated with 5 μL inoculum solution containing ½ strength of potato dextrose broth (PDB-Difco at 12 g/L) and 5 × 10 5 spores/mL.

| Plant growth analysis
Relative growth rate (RGR) was used to analyze tomato growth after chitosan treatment as described . Growth analysis of Arabidopsis plants was performed by measuring rosette perimeter using Photoshop CS5 (Vasseur, Bresson, Wang, Schwab, & Weigel, 2018).

| Callose deposition assays
For analysis of callose deposition after chitosan treatment, material from tomato and Arabidopsis plants with different concentrations of chitosan were collected 1 day after treatment (dat) and placed in 96% (v/v) ethanol in order to destain leaves. Aniline blue was used to stain callose deposits as described previously (Luna et al., 2011). Analysis of callose associated with the infection by B. cinerea in tomato leaves was performed as described (Rejeb, Pastor, Gravel, & Mauch-Mani,-2018)  Infection-associated callose was scored and analyzed in a similar way but callose intensity was expressed relative to fungal lesion diameters.
Image analyses were performed with Photoshop CS5 and ImageJ.

| Chitosan antifungal activity in vitro assay
Botrytis cinerea mycelial growth assessment was performed using Potato Dextrose Agar (PDA) as culture media with different concentrations of chitosan (1%, 0.1%, 0.01% w/v). PDA was autoclaved and then chitosan and the fungicide Switch (as positive fungicide control) (1%, 0.1%, 0.01% w/v) were added directly to PDA as it cooled. One 5 mm diameter agar plug of an actively growing B. cinerea mycelium was added per plate. Five plates per treatment were sealed with parafilm and then incubated under controlled conditions (darkness and 24 C).
After 4 days, the mean growth of the fungus was determined by measuring two perpendicular diameters and calculating the mean diameter.

| High-pressure liquid chromatography (HPLC)mass spectrometry (MS)
Healthy and infected tomato leaf tissues were harvested in liquid nitrogen and subsequently freeze-dried for 3 days. Freeze-dried samples were ground in 15-mL Falcon tubes containing a tungsten ball in a bead beater. Ten milligrams of each sample was used for hormone extraction. Sample extraction, HPLC-MS quantitative analysis of plant hormones and data analysis were performed as described (Forcat, Bennett, Mansfield, & Grant, 2008). Accurate quantification of abscisic acid (ABA), salicylic acid (SA) and JA used the deuterated internal standards added during sample extraction (Forcat et al., 2008) and concentrations were calculated using standard concentration curves. Due to the lack of a standard, relative accumulation of jasmonic acid-isoleucine (JA-Ile) were obtained by calculations of % peak areas among samples. Following Lowess normalization, data were re-imported as singlecolour data. Data were filtered to remove probes that did not have detectable signal in at least three replicates, leaving 22,381 probes for statistical analysis.

| Transcriptome analysis
Analysis of Variance (2-way ANOVA; p-value ≤ .01, Benjamini-Hochberg false discovery rate correction) was used to identify differentially expressed genes (DEGs) for the factors "Treatment" (3,713 DEGs), "Time" (6,920) and "Treatment-Time interaction" (186). Subsequently, pairwise Student's T-tests were performed (Volcano plots: p-value ≤ .05, twofold cut-off) on the global set of 8,471 DEGs for each of the three test treatments (Chitosan + Mock, Water + Mock and Chitosan + B. cinerea) compared to control (Water + Mock) at each time point. Venn diagrams were used at each time point to identify common and specific DEGs.

| Panther gene ontology (GO) term enrichment analysis
Panther software (Thomas et al., 2003) was used to visualize DEG products in the context of biological pathways and/or molecular functions, using default settings. Functional enrichment analysis was performed using DEG lists for Chitosan + B. cinerea and Water + B. cinerea treatments at 6 hpi. "Biological processes" and "molecular functions" were selected using PANTHER Overrepresentation Test (release 20170413) against S. lycopersicum (all genes in database) and Bonferroni correction for multiple testing.

| DEG transcript co-expression analysis
Two-way ANOVA was performed on the filtered microarray dataset at increased stringency (p-value ≤ .01, Bonferroni false discovery rate correction) to identify 1,722 highly significant DEGs. Pearson's correlation was used with default settings in Genespring (v 7.3) to generate a heatmap to help identify co-expressed transcripts ( Figure 3b).

| Gene expression analysis
Validation of S. lycopersicum transcriptomic analysis was performed by qRT-PCR of nine candidate differentially expressed genes (DEGs), comparing gene expression values with microarray. RNA samples were DNAse-treated with TurboDnase (ThermoFisher) and complementary DNA (cDNA) was synthesized from 2.5 μg total RNA using Superscript III reverse transcriptase (Invitrogen) as recommended with random hexamer/oligo dT primers. RT-qPCR reactions were performed with specific S. lycopersicum oligonucleotide primers (Table S4) purchased from Sigma-Aldrich. Gene primers were designed using Universal Probe Library (UPL) assay design centre (Roche Diagnostics Ltd.). RT-qPCR was performed using FastStart Universal Probe Master Mix (Roche) and expression was calculated against two reference genes (SlActin-like and SlUbiquitin) using the Pfaffl method (Pfaffl, 2001).

| Gene cloning
Orthologues of SlACRE75 and SlACRE180 were obtained from CDS and protein sequences BLAST analysis against Arabidopsis genome (TAIR10) for Arabidopsis sequences, or a reciprocal best BLAST hits (RBH) (Ward & Moreno-Hagelsieb, 2014)   These experiments were repeated once.

| Confocal microscopy analysis
For the analysis of the subcellular localization, A. tumefaciens GV3101 carrying plasmids with expression constructs were co-infiltrated with pFlub vector (RFP-peroxisome tagged marker) into leaves of 4-weekold N. benthamiana CB157 (nucleus mRFP marker) and CB172 (ER mRFP marker) reporter lines using 1 mL needleless syringes. Two days after infiltration, leaves were excised and prepared for confocal microscopy. GFP and mRFP fluorescence was examined under Nikon A1R confocal microscope with a water-dipping objective, Nikon X 40/1.0 W. GFP was excited at 488 nm from an argon laser and its emissions were detected between 500 and 530 nm. mRFP was excited at 561 nm from a diode laser, and its emissions were collected between 600 and 630 nm.

| Western blot analysis
Leaves from N. benthamiana leaves infiltrated with A. tumefaciens GV3101 carrying plasmids with expression constructs were excised, ground and proteins extracted, as previously described (Gilroy et al., 2011;Yang et al., 2016). Western blotting was performed as previously described (Qin et al., 2018). Detection of GFP was performed using a polyclonal rabbit anti-GFP antibody (1:4,000 dilution) and secondary anti-mouse antibody (IG HRP 1:10,000) according to the manufacturer's instructions. ECL development kit (Amersham) detection was used according to the manufacturer's instructions.

| Transformation of Arabidopsis thaliana stable over-expression transgenic lines
Arabidopsis over-expression plants were transformed using A. tumefaciens GV3101 carrying plasmids with expression constructs using the flower dipping method (Clough & Bent, 1998). Selection of F I G U R E 1 Characterization of chitosan-induced resistance in tomato and Arabidopsis. (a) Disease lesions in tomato and (b) in Arabidopsis at 3 days post inoculation. Values represent means ± SEM (n = 4-10). (c) Callose deposition triggered by chitosan treatment in tomato and (d) in Arabidopsis leaves 1 day post spray treatment. Values represent means ± SEM (n = 8-10) of % of callose per leaf area. Different letters indicate statistically significant differences among treatments (least significant differences for graph a and Dunnett T3 Post-Hoc test for graphs b, c and d, α = 0.05) Arabidopsis transformants and homozygous lines selection were performed as described (Luna et al., 2014). Resistance was tested against B. cinerea as described before. Two independent homozygous overexpression lines were obtained per construct apart from construct NbACRE75, where only one line was obtained.

| Pathosystem statistics
Statistical analysis of IR and growth phenotypes were performed as described . Data analysis was performed using SPSS Statistics 23 and GenStat ® 18th Edition (VSN International, Hemel Hempstead, UK). Statistical analysis of resistance phenotypes in Arabidopsis over-expression lines was done by ANOVA with "construct" as a single treatment factor at 10 levels and resulting value from the average between both lines per construct: Col-0 (wild-type treatment); two empty vector lines "EV 3.1" and "EV 4.1"; "SlACRE75 1.1" and "SlACRE75 2.1"; "SlACRE180 1.2" and "SlACRE180 3.1"; "NbACRE180 1.1" and "NbACRE180 2.1"; and "NbACRE75 1.  (Figure 1a). The resistance phenotype induced by chitosan had a dose-dependent effect at the two high concentrations (1% and 0.1%); however, the lowest concentration (0.01%) induced a level of resistance in between 0.1% and 1% treatments. In Arabidopsis, chitosan treatment resulted in IR in a concentrationdependent manner, with 1% having the strongest effect (Figure 1b). In aubergine, chitosan treatment resulted in differences in lesion diameter in all concentrations compared to water-treated control plants ( Figure S1), however, post-hoc analysis demonstrated that 0.1% was the most effective concentration.
We then tested whether chitosan induces callose deposition in a similar manner to other chitosan formulations (Luna et al., 2011).
Plants were treated with increasing concentrations of chitosan 1 day before aniline blue staining. In both plant species, treatments with chitosan resulted in a direct induction of callose. The lowest concentrations of 0.001% and 0.01% in tomato and Arabidopsis, respectively, triggered the strongest effect (Figure 1c,d).
To determine any antifungal effect of chitosan, different concentrations were tested on B. cinerea hyphal growth in vitro and compared to different concentrations of the fungicide Switch (Syngenta).
Whereas, all concentrations of Switch arrested pathogen growth, only 0.1% concentration of chitosan or higher had an antifungal effect ( Figure S2). However, the lowest concentration of chitosan tested (0.01%) had no antifungal effect compared to the control. This shows a concentration threshold for chitosan-direct antifungal activity against B. cinerea. Since 0.01% chitosan had no antifungal effect, but reduced B. cinerea lesions and induced callose formation, this concentration was selected for more in-depth analysis.

| Analysis of defence priming mechanisms marking chitosan-induced resistance
We tested whether IR triggered by chitosan is mediated by priming mechanisms through the assessment of its capacity to induced longlasting resistance in distal parts of the plants. Treatments with 1% chitosan induced long-lasting resistance against B. cinerea of at least 2 weeks after initial treatment of tomato plants (Figure 2a).
In order to assess whether treatments with chitosan directly affects plant development, we tested plant growth 1 week after treatment with 1% chitosan. These experiments revealed that chitosan treatment triggers a statistically significant growth promotion, therefore indicating that IR by chitosan does not negatively impact plant development ( Figure S3a).
To study whether chitosan IR was based on known mechanisms

| Transcriptional analysis of chitosan-induced resistance
Priming of gene expression normally follows a characteristic pattern: differential expression is low, transient or often non-detectable after treatment with the elicitor only (i.e., Chitosan + Mock) and enhanced differential expression occurs upon subsequent infection (i.e., Chitosan + B. cinerea) compared to infected plants that were not pretreated with the chemical (i.e., Water + B. cinerea) (Conrath et al., 2006;Martinez-Medina et al., 2016). Importantly, the expression kinetics are also key points for the establishment of defence priming. To further determine the priming basis of chitosan-IR, we performed whole transcriptomic analysis at 6, 9 and 12 h post infection (hpi) with B. cinerea. These time points were selected as they cover the early, non-symptomatic start of the B. cinerea infection process. Unsupervised data analysis was first performed to observe global changes in the experiment. For this, we did a 2D principal component analysis (PCA) at different hours post infection. This analysis shows that chitosan treatments did not trigger major changes in transcription, however, it was the infection with B. cinerea which greatly impacts the experiment (Figure 3a). Moreover, whereas separation can be observed between Mock-and B. cinerea-infected replicates at 9 and 12 hpi, no obvious differences could be seen in the PCA at the  (Figure 3b). Overall patterns aligned with the previous finding that infection with B. cinerea had a large-scale, more extensive and differential response on tomato transcription compared to treatment with chitosan ( Figure 3a). Moreover, the analysis demonstrates that application of chitosan results in a higher number of genes repressed than induced, with the exception of some highly induced genes in cluster iv. Distinct differences were evident between treatment with chitosan compared to infection with B. cinerea, for example, a large group of genes in cluster iv differentially induced by B. cinerea at 9 and 12 h, as well as a large group of genes repressed by the pathogen in cluster i. This indicates that chitosan works as a priming agent that does not directly trigger major effects in gene transcription.  (Table 1). Moreover, for molecular function, cysteine-type peptidase activity, transcription factor activity, sequence-specific DNA binding and nucleic acid binding transcription factor activity were enriched (Table 1).

| Identification of genes primed by chitosan
To identify genes that could be involved in chitosan-IR, gene expression profiles were scrutinized. First, qRT-PCR analysis of a subset of nine genes was done to successfully validate the expression data of the microarray (Figure S4a,b). Similar expression profiles were observed in the microarray and the qRT-PCR data, validating the data set. Priming profiles, that is, subtle or non-detectable differential  (Table S1) and 57 up-regulated (Table S2) genes were found. An overrepresentation test was performed to investigate gene ontology categories of the primed genes (Panther 14.0).
Among the 203 genes that were repressed during infection (Table S1), 11 transcripts were associated with cysteine-type peptidase activity. Other transcripts were grouped with photosynthesis, light harvesting in photosystem I activity. Moreover, several had a response to hormone activity; nine ethylene-responsive transcription factor and receptor genes were significantly down-regulated from −2.3 to −1.1 compared to Water + B. cinerea. Other notable genes with strong priming include those with proteolysis activity, with a range between −3to −1.7-fold repressed. Other genes with repressed expression belong to auxin hormones and one to the ABA receptor (ABAPYL4). Furthermore, two genes of the little-known LAT-ERAL ORGAN BOUNDARIES (LOB) were identified as repressed.
Additional transcripts were functionally unassigned within the list.
Among the 57 differentially up-regulated genes (Table S2), there was one transcript encoding peroxidase activity with twofold increase compared to Water + B. cinerea, nine transcripts encoding protein kinase activity with between +1.1-to +2.1-fold, five transcripts encoding transcription regulatory activity, including SlMYB20, SLWRKY51 and SlWRKY72. Additional transcripts were functionally unassigned within the list.
Importantly, uncharacterized genes also show primed expression patterns. Of these, Avr9/Cf-9 rapidly elicited protein 75 (ACRE75; Solyc11g010250.1) was up-regulated 1.6-fold in Chitosan + B. cinerea in comparison to water + B. cinerea at 6 hpi (Table S2). ACRE genes have been previously studied and characterized as important genes involved in R gene-mediated and ROS gene-independent early plant defence responses (Durrant, Rowland, Piedras, Hammond-Kosack, & Jones, 2000) and in response to methyl-jasmonate (MeJA) treatment (Van Den Burg et al., 2008). ACRE75 molecular functions are still to be deciphered and therefore research into its role and other members of the ACRE gene family in defence priming of chitosan was pursued.

| Role of ACRE genes in induced resistance against Botrytis cinerea
In order to investigate whether other members of the ACRE gene family display a similar priming profile to ACRE75, correlation analysis was performed on the subset of genes differentially expressed at 6 hpi. Genes with statistically significant similar profiles were identified (Table S3), which included ACRE180 at a confidence value of 0.956. In addition, analysis of the samples later in the experiment, confirmed that both ACRE75 and ACRE180 are primed also at later time points (Figure S4a,b).
In order to investigate whether primed expression of ACRE75 and ACRE180 genes may be involved in enhanced disease resistance, genes from S. lycopersium and orthologue genes in N. benthamiana were overexpressed using both transient and stable systems. For SlACRE75, best match against N. benthamiana genome was Niben101Scf03108g12002.1 (termed NbACRE75), sharing a 77.5% protein identity; (ii) For SlACRE180, the best match against the N. benthamiana genome was Niben101Scf12017g01005.1 (termed NbACRE180), with 49.5% protein identity. Arabidopsis orthologue analysis failed to identify hits for ACRE75 and ACRE1280 candidate genes. Constructs were produced with a fused-GFP protein in the N-terminus and protein integrity was confirmed via Western blot.
Proteins extracted from N. benthamiana leaves 48 h after agroinfiltration and Western blot analysis confirmed that they were the expected sizes ( Figure S5). Subcellular location of proteins was analyzed via confocal microscopy of GFP fluorescence. Over-expression constructs were co-infiltrated with RFP-marker pFlub vector T A B L E 1 Biological processes and molecular functions of enriched genes  Foliar applications of chitosan have been widely used to control disease development caused by numerous pests and pathogens (El Hadrami et al., 2010). However, few studies have investigated the role of chitosan as a priming agent and most have focused on its use as a seed priming elicitor mainly to improve germination and yield (Guan, Hu, Wang, & Shao, 2009;Hameed, Sheikh, Farooq, Basra, & Jamil, 2013). Here, we show that chitosan-IR is based on priming of defence mechanisms. Our experiments confirmed that chitosan-IR is not associated with growth reduction ( Figure S3a), was durable and maintained for at least 2 weeks after treatment (Figure 2a), and that is based on a stronger accumulation of callose at the site of attack and accumulation of JA ( Figure 2d) and JA-ile ( Figure S3b). These results demonstrate that fungal growth arrest after chitosan treatment is not directly mediated by the toxicity effect of the chemical, as the infected leaves were formed after treatment and therefore were not sprayed with the elicitor. Moreover, these results demonstrate similar priming mechanisms after chitosan treatment to other elicitors, includ- In order to further explore priming of defence and to unravel the transcriptional mechanisms behind chitosan-IR, we performed transcriptome analysis. In our experiment, using a concentration of chitosan that is associated with defence priming but with no direct antimicrobial effect, we identified early acting differential trans- Panther enrichment analysis showed that at 6 hpi, the number of down-regulated DEGS was more than three times up-regulated ones Transcriptomic (Table S2 and Figure S4a) and qRT-PCR ( Figure S4b) analyses showed that chitosan can prime ACRE75 for a faster and stronger expression after infection with B. cinerea. ACRE genes have been linked to plant defence responses. Similar genes were previously identified in tobacco cells to exhibit rapid Cf-9dependent change in expression through gene-for-gene interaction between the biotroph pathogen Cladosporium fulvum avirulence gene (Avr9) and tomato resistance Cf-9 gene (Durrant et al., 2000). To determine the role of ACRE genes in priming by chitosan, we searched for other ACRE genes showing similar expression profiles to ACRE75 and this revealed that ACRE180 displays a similar priming profile. This was more evident at 9 hpi ( Figure S4b) than at 6 hpi, suggesting that the role of ACRE180 is later time than ACRE75. Subcellular localisation may indicate why priming of these genes does not occur at the same time; whereas ACRE75 accumulates exclusively in the nucleus and nucleolus ( Figure S6e Therefore, our results confirm involvement of ACRE genes in plant immunity and suggest an involvement in chitosan-induced priming due to their expression profiles. Interestingly, the IR effect was greater in Arabidopsis plants over-expressing ACRE75 in comparison to ACRE180 (Figure 4c), which could corroborate our evidence of earlier activity of ACRE75, therefore being more effective during early resistance response. More work in needed to unravel the molecular function of ACRE75 and ACRE180 in the expression of defence mechanisms. Whereas, it is unlikely that the over-expression of ACRE75 and ACRE180 trigger the constitutive activation of defence mechanisms due to the lack of reduced growth phenotypes ( Figure S7), future work will study whether these lines are constitutively primed to express defence mechanisms. Nevertheless, fine-tuning of priming-