Cloning and differential expression of a plum single repeat-myb, PdMYB3, in compatible and incompatible interactions during fungal infection

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repeat-MYB,

PdMYB3

, in compatible and incompatible

interactions during fungal infection

Ashraf El-kereamy

2

and Subramanian Jayasankar

1,3

1_{University of Guelph, Department of Plant Agriculture. 4890 Victoria Ave. N., P. O. Box 7000 Vineland Station,}

Ontario, Canada L0R 2E0; and2Horticulture Department, Faculty of Agriculture, Ain Shams University, Cairo, Egypt.Received 12 January 2013, accepted 1 March 2013.

El-kereamy, A. and Jayasankar, S. 2013. Cloning and differential expression of a plum single repeat-MYB, PdMYB3, in compatible and incompatible interactions during fungal infection. Can. J. Plant Sci. 93: 599605. Enhancing resistance to pathogen attack through conventional breeding is a major challenge, especially in perennial species. Monilinia fructicola fungal infection causes brown rot disease, resulting in economic damage of stone fruits at flowering, pre- and post-harvest stages. The molecular mechanism of resistance to this disease is still not known. In the present study, we cloned and analyzed the expression of a novel MYB transcription factor from European plums (PdMYB3) induced in response to

M. fructicola fungal infection. The identified PdMYB3 is a single repeat-MYB protein that contains a conserved

SHAQKYF motif. Monilinia fructicola infection induces the expression of PdMYB3 in fruits of four cultivars within 24 h; however, it is differentially expressed in the susceptible and resistant varieties. By comparing four different cultivars we found that PdMYB3 is induced in much higher levels in the susceptible cultivars than the resistant ones. In addition the

PdMYB3expression is higher in the early stages of fruit development prior to pit hardening, suggesting a potential role for

PdMYB3during this stage. Promoter analysis revealed the presence of some hormone cis-elements suggesting a possible

role for PdMYB3 gene in transmitting a signal from the hormonal pathways to downstream components during host-pathogen interactions.

Key words: Plum, fruits, MYB, brown rot

El-kereamy, A. et Jayasankar, S. 2013. Clonage et expression diffe´rentielle du facteur de transcription MYB microsatellitaire PdMYB3 du prunier chez les cultivars compatibles et incompatibles lors d’une infection par les cryptogames. Can. J. Plant Sci. 93: 599605. Accroıˆtre la re´sistance aux agents pathoge`nes par les me´thodes classiques d’hybridation soule`ve de grandes difficulte´s, surtout avec les espe`ces vivaces. L’infection par Monilinia fructicola engendre la pourriture brune a` l’origine des pertes ećonomiques enregistreés chez les fruits a` noyau au stade de la floraison ainsi qu’avant et apre`s la cueillette. On ignore toujours le mećanisme molećulaire a` la base de la re´sistance a` cette maladie. Les auteurs ont clone´ puis e´tudie´ l’expression d’un nouveau facteur de transcription MYB issu de pruniers europeéns (PdMYB3), consećutivement a` l’infection causeé par M. fructicola. PdMYB3 est une proteíne microsatellitaire de type MYB dans laquelle a e´te´ conserve´ le motif SHAQKYF. L’infection par M. fructicola a dećlenche´ l’expression de PdMYB3 dans les fruits de quatre cultivars en l’espace de 24 heures; neánmoins, l’expression du facteur de transcription n’e´tait pas la meˆme chez les varie´te´s sensibles et re´sistantes. En comparant quatre cultivars, les auteurs ont constate´ l’induction de PdMYB3 a` une concentration beaucoup plus e´leveé chez les varie´te´s sensibles que les re´sistantes. De plus, PdMYB3 s’exprime plus fortement aux premiers stades du de´veloppement du fruit, soit avant le durcissement du noyau, signe que PdMYB3 pourrait jouer un roˆle sur ce plan. L’analyse des promoteurs re´ve`le l’existence de quelques e´le´ments hormonaux cis, ce qui pourrait signifier que le ge`ne

PdMYB3 transmet un signal hormonal a` des composants en amont lorsqu’il y a interaction entre l’hoˆte et l’agent

pathoge`ne.

Mots cle´s: Prune, fruits, MYB, pourriture brune

Upon pathogen infection, plants initiate a large spec-trum of defense mechanisms. Plants respond to patho-gen signals with several defense responses such as cell lignification and activation of pathogen-related proteins to limit invasion of fungal hyphae (Showalter et al. 1985). As a part of signal transduction due to pathogen attack, the host cells induce a number of transcription factors. These transcription factors activate the

tran-scription of the biosynthetic genes involved in a myriad of defense responses.

The MYB family of transcription factors regulates numerous processes during the plant life cycle and responses to environmental stress (Smolen et al. 2002). MYB proteins are classified into three major groups based on the number of adjacent repeats in the binding domain; R1R2R3-MYB, R2R3-MYB, and R1-MYB.

3

Corresponding author (e-mail: [email protected]). Abbreviation: PCD, programmed cell death

Can. J. Plant Sci. (2013) 93: 599605 doi:10.4141/CJPS2013-009 599

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Most plant MYB proteins are of R2R3 type (Jin and Martin 1999; Stracke et al. 2001; Du et al. 2009). The plant R2R3-MYB is involved in a range of physiological responses such as regulation of the isopropanoid and flavonoid pathway (Mellway et al. 2009; Lin-Wang et al. 2010) and various other defense and stress responses (Yang and Klessig 1996; Sugimoto et al. 2000; Lee et al. 2001; Raffaele et al. 2008). In addition to the conserved adjacent repeats, other conserved domains have been reported in some of the plant MYB proteins (Li et al. 2006). Mercy et al. (2003) reported the isolation and cloning of a single MYB-repeat maize protein with a motif that consists of VASHAQKYF amino acids.

A number of the MYB transcription factors are involved in plant defense to pathogen infection. For example, the tobacco mosaic virus (TMV)-inducible myb (MYB1) is associated with hypersensitivity and the development of systemic acquired resistance in resistant cultivars (Yang and Kleesig 1996). The rice jasmonic acid-inducible JAmyb is associated with infection by blast fungus (Pyricularia grisea) and its transcription is higher in the susceptible than the resistance interaction (Lee et al. 2001). Further, Arabidopsis AtMYB30 acts as a positive regulator for the hypersensitive response leading to cell death during pathogen infection (Vailleau et al. 2002). Although several members of MYB family have been involved in development and osmotic stress of fruit species (Feng et al. 2006; Espley et al. 2007; Czemmel et al. 2009; Mahjoub et al. 2009; Lin-Wang et al. 2010; Gao et al. 2011), their role in stone fruit defense responses against fungal infection, has not been studied yet.

European plum (Prunus domestica L.) is a commer-cially important stone fruit species. Like other fruit crops, these plums are affected by several pathogenic microbes during ripening, causing huge economic losses around the world. One of the major diseases of European plum is brown rot, caused by the fungus Monilinia fructicola, which attacks the fruits as they begin ripening, and continues to be detrimental through post-harvest storage and shipping. Infection starts from latent spores on the trees or from mummified fruits. The disease starts as small lesions on the skin of the fruits, which grow as the infection progresses. Lesions can cover the whole fruit surface, effectively mummifying it. Infection can also occur during fruit storage. In addition, this fungus causes severe blossom blight. All stone fruits are vulner-able to this disease and exhibit similar symptoms and damage. Understanding the resistance mechanisms to brown rot disease at the molecular level could be functionally translated to successful cultivars. To achieve this goal, a number of the transcription factors were cloned from the infected fruits, including members of the MYB family, known for their involvement in the response to environmental stress. Here, we focused on the PdMYB3 which show differential expression after M. fructicolainfection.

MATERIALS AND METHODS Plant Materials and Fungal Inoculation

Fruits used in this study were collected from mature trees maintained at the Experimental Station, University of Guelph, Vineland, Ontario, Canada. For in vitro fungal inoculation, plum fruits were harvested at commercial maturity and inoculated with 20mL of conidial suspen-sion (1 103 conidia mL1) or 20 mL of water (mock inoculation). The inoculation was carried out by punch-ing one 0.2-mm hole into the fruit and insertpunch-ing the conidial suspension or the water using a pipette. After inoculation, the fruits were incubated at room tempera-ture in clear tote boxes with lids. Sterilized paper towels drenched with sterile water were placed inside the totes to increase humidity and favor fungal growth. Skin and flesh of approximately the same size were carefully excised from the infected area daily, and up to 4 d from eight fruits pooled together for one replicate then frozen immediately in liquid nitrogen. For each study, three such biological replicates were used. In addition to the artificial infection, samples from healthy and natu-rally infected fruits were collected from the same trees. Each replicate contained three infected fruits and three replicates were used for analysis. The natural infection was confirmed by laboratory analysis of the fungi. All frozen materials were stored at 808C until further analysis.

RNA Extraction and cDNA Synthesis

Fruit tissues were ground in liquid nitrogen using a 6750 SPEX Sampleprep Freezer/Mill (SPEX Certiprep, Metuchen, NJ). Total RNA was extracted from 2 g of fruit tissues, as described by Boss et al. (1996). DNA was removed from the samples using the RNase free DNaseI treatment according to the manufacturer’s instructions (Promega, Madison, WI) followed by a cleanup with RNeasy mini kit (Qiagen, Mississauga, ON). Two micrograms of DNase-treated RNA was used to synthesize the first strand cDNA using oligo-dt primers and M-MuLV reverse transcriptase (New England Biolabs, Vancouver, BC) according to manu-facturer’s instructions.

Cloning ofPdMYB3Gene

First strand reaction product from the cDNA ob-tained above was used to clone a full-length PdMYB3 gene from plums. The PCR primers (A69MybF3, GGGCGCTCCAGCCTATTCGAC and A70MybR3, GACACACAGAGAGGCCACAAGG) were designed using the available EST sequence from peach. Based on the sequence of primary fragment, other primers were designed and used to clone the full-length cDNA. For sequencing, the PCR product was cloned into a pGEMT easy vector (Promega Madison, WI). The full-length cDNA has been submitted to the Genebank database under the accession number (KC427983). Plum genomic DNA was isolated from immature leaves using the

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CTAB method and used to clone the promoter region of the PdMYB3 by using the Universal Genome Walker Kit (Clontech, Palo Alto, CA) according to the manu-facturer’s guidelines. All the sequencing in this study was carried out by Macrogen, Seoul, S. Korea.

PdMYB3 Recombinant Protein Production in E. coli

For heterologous expression of the PdMYB3, we cloned the full-length cDNA sequence into the protein expres-sion vector PET-28a (Novagen, WV) in the EcoR1 cloning site using the forward primer GAATTCAT-GACTCGGCGGTGCTCGCAC and the reverse pri-mer GAATTCGACTGCTTGGATCGCACTGCTCT. The ligation product was cloned into E. coli BL21 followed by sequencing the positive clones to confirm the orientation and the peptide reading frame. The bacteria were grown in LB medium, supplemented with antibiotics, at 378C until A600 0.60.7. Expression of the recombinant protein was induced with 1 mM IPTG. His-Tag fusion protein purification was carried out under denatured conditions using Ni-NTA agarose bead. After centrifuging, cells were resuspended in the lysis buffer (6 M urea, 50 mM Na2HPO4pH 8.0, 0.3 M

NaCl and 1 mM PMSF) and sonicated on ice for 20 s three times. The crude extract was collected after centrifugation for 15 min at 48C. Ni-NTA agarose beads (Qiagen) were equilibrated using a buffer containing 6 M Urea, 50 mM Na2HPO4 pH 8.0 and 0.5 M NaCl

and incubated with crude extract for 1 h at 48C. After incubation, the beads were washed three times using the same buffer and the protein was eluted using the elution buffer (6 to 8 M urea, 20 mM Tris pH 7.5, 100 mM NaCl, and 250 mM imidazole). The purified protein was analyzed by separating on SDS-PAGE gel.

Quantitative RT-PCR

To study the transcription level of the PdMYB3 in different fruit tissues, quantitative real-time PCRs were performed using the equivalent of 5 ng of total RNA in a 20-mL total reaction volume using 0.75 cyber green fluorescence. All QRT-PCR experiments were run in three biological replicates. Relative fold differences were calculated based on the comparative Ct method using actin of respective species as an internal standard and the 2DDCt formula. We used the PdMYB3F forward primer (AGTGCAGGGCCCATGAGAGAATTA) and PdMYB3R primer (CATGCCCACCAGTGCATCAA-CATT) as a reverse primer to perform the Real-Time PCRs. Actin primers were used for internal control as described in El-kereamy et al. (2011).

RESULTS AND DISCUSSION

Cloning and Sequence Analysis of the PdMYB3

To identify the transcription factors involved in the plums’ response to M. fructicola infection, several trans-cription factors were cloned from the infected fruits,

and expression analysis showed the potentiality of the PdMYB3 in this response. In this study we show the cloning of a full-length cDNA sequence encoding a plum MYB transcription factor (PdMYB3) involved in plum fruit and brown rot interactions. Sequence analy-sis of the full-length PdMYB3 cDNA revealed that it comprises 1071 bp (357 amino acids). The derived amino acid sequence indicates the presence of a VASHAQKYF amino acid segment conserved among homologous pro-teins (Fig. 1). This signature motif is present in several other plant species including Arabidopsis and maize as well as the microbe Entamoeba histolytica (Mercy et al. 2003; Ehrenkaufer et al. 2009).

The predicted molecular weight of the complete protein was 38.524 kDa. This was further confirmed by heterologous expression of the protein in E. coli cells as shown in Fig. 2A. The PdMYB3 is predicted to be a basic protein as its isoelectric point (IEP) is 8.8. Sequence analysis of 1247 bp of PdMYB3 promoter revealed the presence of several cis-elements involved in hormones signaling such as the ABA responsive elements (ABRE) and GA responsive elements (GARE) in addition to the fungal elicitor responsive element (W-1) and two MYB binding sites (Fig. 2B). The presence of such cis-elements in the promoter suggested the importance of this gene in the hormone signaling pathway. Further the W-1 box involved in the fungal response reinforces the involve-ment of this gene in the plant defense response. Having the MYB binding site PdMYB3 promoter indicates that this gene can be under the activation of another member of the same MYB family. PdMYB3 shares high sequence similarity with previously reported single domain MYB transcription factors identified in other species such as Malus domestica, Genbank accession Number ADL36774 (88%), Medicago truncatula, ABR28340 (77%), Populus trichocarpa, XP_002303311 (75%), Vitis vinifera, XP_ 002283785 (68%), Glycine max, ABH02830 (67%), Ricinus communis, XP_002515966 (64%) and Arabidop-sis thaliana, AAB63650 (51%) (Fig. 3). Despite the high similarity between the PdMYB3 and the above-mentioned orthologs, not much is known about any of them. This high similarity indicates the potential of those orthologs to be involved in the defense response during the pathogen attack.

PdMYB3 MTRRCSHRSNNGHNSRTCPTRAGSSSSSSVGGGGGGGLKL FGVRLTDGSIIKKSASMGNLSSAAHYHSSSPNPDSPSSDL HDPVHVPEGYLSDDPAHASSSANRRGDRKKGTPWTEEEHR MFLIGLQKLGKGDWRGIARNYVTTRTPTQVASHAQKYF IR QSNATRRKRRSSLFDMVPDMAMDPPPVPEEQVFLPSCQ EA ESEAASSLPSLNLSLSSECKPMETTHEEKVKEPDHEPVMG SNGFPPMIPGFFPAYLPYPFPVWPPSAGPMRELKGGEASH QQVLRPIPILRKEPVNVDALVGMSQLSLGDTERGHKEPSP LSLKLLGEPSRQSAFHPNAPAGEPDLSKGKSSAIQAV*

Fig. 1. Amino acid sequence of the plum PdMYB3; the conserved domain (VASHAQKYF) is underlined.

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Differential Expression ofPdMYB3in Susceptible and Resistance Interactions with M. fructicola

To study the involvement of PdMYB3 in response to brown rot disease, we analyzed its transcript accumula-tion in four European plum cultivars with varying degrees of resistance to brown rot disease. ‘Stanley’ and ‘Violette’ are less susceptible cultivars than ‘Veeblue’ and ‘Victory’. Quantitative real time PCR analysis re-vealed that PdMYB3 transcript level is generally induced in all the four varieties within 24 h of M. fructicola infection and continued up to 4 d (Fig. 4). However, this induction was higher in the susceptible cultivars, ‘Veeblue’ and ‘Victory’ reaching 19.7 (92.2) and 20.1 (90.44) compared with 12.4 (91.1) and 11.1 (94.1) in the resistant varieties, ‘Stanley’ and ‘Violette’ 3 d post-inoculation. The same trend was observed after 4 d reaching 19 (90.15) and 34 (92) susceptible cultivars compared with 11.3 (90.15) and 3.7 (92.4) in the resistant varieties (Fig. 4). To confirm these results, we collected naturally infected fruits from the trees and analysed the expression of PdMYB3 in those fruits.

We found that the transcript levels of PdMYB3 follow the same trend as observed among in vitro infected fruits (Fig. 5). In the naturally infected fruits, PdMYB3 transcription level was 425 (951) and 330 (921) in the susceptible cultivars, ‘Veeblue’ and ‘Victory’ compared with 244 (924) and 86 (915) in the resistant varieties, ‘Stanley’ and ‘Violette’. The association of the higher Fig. 2. Puriﬁcation and promoter analysis of PdMYB3.

Full-length cDNA sequence was cloned in the expression vector Pet28a and the protein was expressed in E. coli, protein was puriﬁed under denatured condition as described in the Materials and Methods section. The Coomassie Blue stain of the elute shows bands of the expected sizes on 12.5%

SDS-polyacrylamide gel. Lane 1 and 2 indicate 4 and 20mg of

PdMYB3 recombinant protein (A). Potential cis-elements presented in the PdMYB3 promoter sequence (B), GARE, cis-acting regulatory element involved in GA-responsiveness; W-1 box, fungal elicitor responsive element; ABRE, cis-acting element involved in abscisic acid responsiveness and MBS, MYB binding site. The numbers below the diagram indicate the position of each cis-element upstream of the ATG initiation codons. PdMYB3 MdMYBR PtMYB RcMYB MtMYB52 GmMYB62 VvMYB AtMybSt1 0.05

Fig. 3. Phylogenetic analysis of PdMYB3 sequence shows the relationship between PdMYB3 and its orthologs in the other plant species. A phylogenetic tree was drawn using Tree View based on alignments by CLUSTALX. The amino acids se-quences used in this analysis are from Prunus domestica PdMYB3 Genbank accession Number, KC427983; Malus domestica, ADL36774; Medicago truncatula, ABR28340; Populus tricho-carpa, XP_002303311; Vitis vinifera, XP_002283785; Glycine max, ABH02830; Ricinus communis, XP_002515966 and Arabidopsis thaliana, AAB63650.

0 5 10 15 20 25 30 35 40

Days post infection (DPI)

0 1 2 3 4

PdMYB3 transcripts level

(Fold change)

Stanley VeeBlue Victory Violette

Fig. 4. Changes in the transcripts level of the PdMYB3 in four European plum varieties 0, 1, 2, 3 and 4 d after the infection

with Monilinia fructicola conidial suspension (1 103_conidia

mL1_{). ‘Stanley’ and ‘Violette’ are more resistance to}

Mon-ilinia fructicolathan ‘Victory’ and ‘Veeblue’.

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PdMYB3 expression with the susceptibility to the M. fructicolainfection suggests a possible role for this gene in programmed cell death (PCD). It seems that the cells of these susceptible varieties ‘Veeblue’ and ‘Victory’ have a different response than less susceptible varieties ‘Stanley’ and ‘Violette’ that might involve the PdMYB3 causing the cells to be more sensitive to pathogen in-fection. In general, the host plant activates R genes as a defense response in a non-compatible interaction caus-ing an inhibition of pathogen development in the host tissues. The compatible interaction of susceptible vari-eties probably involves alteration of R gene transcripts to low levels allowing the pathogen to grow within the host cell. Cells try to stop pathogen progression by PCD surrounding the infection site. This PCD is essential for activating the plant cell responses to the pathogen attack and required the presence of several genes to activate the hypersensitivity responses (HR) and activate the defence mechanism pathway leading to more resistance against the pathogen (Patel et al. 2006). Some genes are reported to be affecting the PCD through the modulation of the inhibitors or the initiators of PCD such as Adi3 gene in tomato (Devarenne et al. 2006). Further, the Arabidopsis AtMYB30was reported to be involved in the cell death program during bacterial infection (Vailleau et al. 2002). Since PdMYB3 expression correlates well with the lesion diameter previously reported in El-kereamy et al. (2011), it is possible that this gene is involved in the regulation of PCD through the activation of PCD inhibitors as observed in tomato plants (Devarenne and Martin 2007). Similar results have been reported during the compatible interaction between Fusarium oxysporum sp. linii and a susceptible flax (Linum usitatissimum) cells (Hano et al. 2008). MYB transcription factors are known to interact with plant hormones (Yanhui et al. 2006). Such inter-actions are often correlated to binding elements in the promoter. The promoter sequence of the PdMYB3

contains elements that bind to plant hormones such as the ABA and GA responsive elements, which are involved in the PCD. It seems that PdMYB3 transcrip-tion could be modulated by the different plant hormones in a similar fashion to other members of the MYB family (Yanhui et al. 2006). Further, plant hormones ABA and salicylic acid are known for their involvement in defense responses during the fungal infection (Adie et al. 2007; Xiong and Yang 2003) and ABA is involved in control-ling PCD in cereals (Guo and Ho 2008). PdMYB3 may play a role in receiving the signals from the ABA and salicylic acid pathways to control the PCD system during fungal infection (Guo and Ho 2008). Several lines of evidences point to the involvement of plant hormones in controlling the expression of PR proteins (Adie et al. 2007). For example, the transcription of the pathogen related protein, PR10 is induced by several stimuli such as methyl jasmonate (MeJA), salicylic acid (SA), gibber-ellic acid (GA3), hydrogen peroxide (H2O2), sodium

chloride (NaCl) and ethylene in different plant species (Agrawal et al. 2001; Xiong and Yang 2003; and Liu et al. 2006). PdMYB3 might play an intermediate role by activating the transcription of some defense-related genes after being activated by hormones during M. fructicolainfection.

Expression ofPdMYB3During Fruit Development Transcripts levels of PdMYB3 in plum fruits at different developmental stages were determined to verify whether PdMYB3 induction is ripening-specific. In all four cultivars the PdMYB3 transcript levels were higher in early fruit development stage, S2 (before pit hardening); however, its transcript levels dropped and remained Stanley VeeBlue Victory Violette

PdMyb3 transcripts level (Fold change) ₀ 2 100 200 300 400 500 _HF NIF

Fig. 5. Induction of the PdMYB3 transcripts under natural infection with Monilinia fructicola in the resistant (‘Stanley’ and ‘Violette’) and susceptible (‘Victory’ and ‘Veeblue’) varieties. HF healthy fruits and NIF naturally infected fruits collected from the trees.

Stanley VeeBlue Victory Violette

Transcript level (Fold change)

0 2 4 6 8 10 S2 S3 S4 S5

Fig. 6. Accumulation of the transcript levels of PdMYB3 during fruit maturation in four European plum varieties. S2 before pit hardening; S3 after pit hardening; S4 full mature stage and S5 ripe stage.

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unchanged in all cultivars and in the following stages: after pit hardening (S3), mature green stage (S4) and ripening stage (S5) (Fig. 6). Accumulation of PdMYB3 transcripts in the early fruit development stage suggests that there could be a potential role for this gene in early development and also that this is not a ripening-associated gene. More studies are required to elucidate the role of the PdMYB3 during embryo and seed develop-ment. It is very unlikely that it is a ripening-related gene, since its transcripts remain stable after the pit hardening until harvest. During this stage of development, fruits are characterized by higher cell division and enlarge-ment, which required higher hormonal level. The higher level of PdMYb3 during this stage indicates the pos-sibility of its involvement in the signal transduction pathways controlling these processes. Previous studies suggested the involvement of the R2R3-MYB genes in the signal transduction pathways of many plant hor-mones such as salicylic acid (Raffaele et al. 2006), abscisic acid (Abe et al. 2003), gibberellic acid (Gocal et al. 1999; Murray et al. 2006), and jasmonic acid (Lee et al. 2001). The induction of the PdMYB3 during fungal infection and its possible role in the resistance to pathogen attack is consistent with role of the several members of the MYB family in the tolerance to different environmental stress conditions. For example, overexpression of the rice OsMYB4 increased the Arabidopsis tolerance to low temperature (Vannini et al. 2004). The loss of Arabi-dopsis AtMYB68 reduces the ability of myb68 mutant plants to compensate their growth at higher tempera-tures (Feng et al. 2004). Further, the BcMYB1 isolated from Boea crassifolia and AtMYB60 isolated from Arabidopsiswere strongly modulated by drought stress and as well (Chen et al. 2005; Cominelli et al. 2005). In the present study, we report for the first time the cloning and the induction of the PdMYB3 during the M. fructicola infection, the pathogen responsible for the brown rot disease in stone fruits.

Identification of PdMYb3 is the first step in under-standing the molecular mechanisms of the susceptibility to the necrotrophic fungus M. fructicola in stone fruits. One could assume that the identified PdMYB3 activates downstream genes involved in the defense responses especially in the susceptible varieties during the M. fructicola infection. One of those target genes might be PR10, which is shown to be induced more in the susceptible variety ‘Veeblue’ compared with the resistant variety ‘Violette’ (El-kereamy et al. 2009). Further work is needed to confirm this hypothesis and elucidate the pathway in which the PdMYB3 is involved.

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