Patent Publication Number: US-11028406-B2

Title: Podosphaera leucotricha resistance providing genes in Malus domestica

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is the United States national phase of International Application No. PCT/EP2016/073783 filed Oct. 5, 2016, and claims priority to International Application No. PCT/EP2015/073135 filed Oct. 7, 2015, the disclosures of which are hereby incorporated in their entirety by reference. 
     The Sequence Listing associated with this application is filed in electronic format via EFS-Web and is hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 1802895_ST25.txt. The size of the text file is 10,606 bytes, and the text file was created on Mar. 28, 2018. 
     The present invention relates to  Podosphaera leucotricha  resistance conferring genes, plants, plant parts and seeds comprising the present resistance providing genes and the use thereof for selecting  Podosphaera leucotricha  resistant plants. 
     Powdery mildew (PM) is a major fungal disease for thousands of plant species, including cultivated Rosaceae such as apple ( Malus domestica ), apricot ( Prunus armeniaca ), peach ( Prunus persica ), and strawberry ( Fragaria  x  ananassa ). Powdery mildew occurs in all major growing regions of Rosaceous crops, leading to severe losses. 
     Apple powdery mildew caused by the fungus  P. leucotricha , is one of the most economically relevant diseases of apple, worldwide. The symptoms are white spots on young green tissues, particularly leaves in the first days after opening, whereas mature leaves show some resistance. Infected leaves crinkle, curl, and prematurely drop. Blossoms and fruits are not the primary targets PM fungi, but infections of these tissues are possible. Powdery mildew spores overwinter in buds and then in spring, with the reprise of vegetative growth, they start a new infection. PM of apple occurs in all apple-producing regions of the world. The disease causes economic damage by reducing tree vigor, flower bud production, and fruit quality. PM of apple produces symptoms on young shoots, leaves, blossoms, and fruit. In general, symptoms are most noticeable on the leaves and fruit. 
       P. leucotricha  is an ascomycete fungus of the  Erysiphaceae  family. During the growing season, this obligate biotroph continuously produces asexual spores (conidia) on specialized short stalks called conidiophores and sexual spores (ascospores) in sac-like asci enclosed in fruiting bodies (ascocarps). Conidia are hyaline (clear, without color) and contain distinct fibrosin bodies, whereas each ascocarp (black) contains a single ascus with eight elliptical ascospores densely grouped together. The hyaline&#39;s fibrosin bodies are refractive inclusion bodies that exhibit varied shapes including rods and cones. Ascocarps do not play any known role in initiating new epidemics, as the ascospores fail to germinate readily. Conidia are wind-dispersed and do not require free moisture to germinate. If they land on susceptible tissue, they initiate infection and produce mycelium. 
     In commercial orchards, fungicides are almost always used to control mildew, as well as other apple diseases. However  P. leucotricha  has exhibited the ability to develop resistance to these fungicides when they are used repeatedly. Benzimidazoles was activity against  P. leucotricha , but their utility in apple disease management is now limited, due to widespread resistance development in  Venturia inaequalis  (apple scab). Fungicides are usually applied at 7- to 10-day intervals from the tight-cluster stage until terminal shoot growth ends to ensure that the application coincides with rapid leaf development and the post-bloom period, and that the new growth do not remain unprotected for long. Furthermore, the intense application of fungicides has several drawbacks. First of all, the effects on the environment are well documented. Secondly, the costs of the chemicals and their applications can reach up to 20% of the total expenses for apple production in some areas. Thirdly, the development of resistant populations of the pathogen was already documented by Baudoin et al. (2008) and Dufour et al. (2011), strongly reducing the efficacy of chemical treatments. Therefore, there is increasing interest in the development of new alternative methods to control PM. 
     The generation of PM-resistant varieties is one of the best options to make sustainable apple cultivation a realistic possibility, preserving at the same time the incomes of the growers. The use of less susceptible apple cultivars may be the most effective means of preventing PM. Apple cultivars, including ‘Jonafree’, ‘Prima’ and ‘Enterprise’, demonstrate natural resistance to PM, but they are not widely cultivated. Cultivar selection is influenced more by commercial appeal, fruit qualities, marketability, and pollination characteristics than by disease resistance. Apple cultivars such as ‘Golden Delicious’, ‘Idared’ and ‘Granny Smith’, are widely grown, but are highly susceptible to PM and require chemical disease management. 
     The most common strategy to develop resistant plants is focused on the introgression of resistance genes (R-genes). R-genes encode proteins that recognize pathogen effectors and trigger defense response, mediated by a signaling network in which plant hormones play a major role (Pavan et al., 2010). Resistance is manifested as localized hypersensitive response at the site of infection (Robert-Seilaniantz et al., 2007; Bruce and Pickett, 2007; Bari and Jones, 2009). R-genes are scarcely durable, as mutations of pathogen effectors, allow to overcome resistance (Parlevliet et al., 1993). Furthermore, R-genes often originate from wild-relatives of the cultivated species, and thus interspecific crossability barriers could prevent their introgression. Moreover, in case of a successful cross, several undesirable traits are incorporated with the R-gene, making extensive backcrossing necessary, which is time-consuming in woody species like apple. 
     An alternative approach is based on the inactivation of susceptibility genes (S-genes), defined as genes whose loss-of-function results in recessively inherited resistance (Pavan et al., 2010). Some pathogens are able to suppress plant defense by activating plant proteins which function is the negative regulation of plant immunity system. The genes encoding these plant proteins are known as susceptibility genes (S-genes) and their knock-out release the suppression of plant defense and lead to resistance (Pavan et al., 2010). The disadvantage of S-genes is the pleiotropic phenotypes sometimes associated to their knock-out (Pavan et al. 2011). Mildew Locus O (MLO) genes are a typical example of PM S-genes. 
     Resistance due to the knock-out of an MLO gene (mlo resistance) was discovered in barley in 1992 (Jorgensen, 1992) and for a long time was considered as a unique form of resistance. However, further studies revealed that MLO genes are largely conserved across plant kingdom and their loss-of-function resulted in resistance in several species, such as  Arabidopsis  (Consonni et al., 2006), pea (Pavan et al., 2011), tomato (Bai et al., 2008) and pepper (Zheng et al., 2013). Not all MLO genes are S-genes and MLO family members are divided in seven clades (Acevedo-Garcia et al., 2014; Pessina et al., 2014). Only two clades contain S-genes: clade IV contains all monocots S-genes (Panstruga et al., 2005; Reinstädler et al., 2010); and clade V contains all dicots S-genes (Consonni et al., 2006; Bai et al., 2008; Feechan et al., 2008; Winterhagen et al., 2008). Not all the members of clades IV and V are S-genes. 
     Considering the economic impact of a  P. leucotricha  infection on apple production, there is a continuos need in the art for  P. leucotricha  resistance providing genes. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention, amongst other objects, to meet this need of the art. 
     According to the present invention, the above object, amongst other objects is met by providing impaired  P. leucotricha  resistance providing genes as outlined in the appended claims. 
     Specifically, the above object, amongst other objects, is met according to a first aspect of the present invention by providing  P. leucotricha  resistance conferring genes, wherein the amino acid sequence encoded by the resistance conferring gene is the primary amino acid sequence represented by SEQ ID No. 1, or a primary amino acid sequence with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity with SEQ ID No. 1 under the condition that the present resistance conferring genes are impaired. 
     Sequence identity as used herein is defined as the number of identical consecutive aligned nucleotides, or amino acids, over the full length of the present sequences divided by the number of nucleotides, or amino acids, of the full length of the present sequences and multiplied by 100%. For example, a sequence with 80% identity to SEQ ID No. 1 comprises over the total length of 589 amino acids of SEQ ID No. 1, 471 or 472 identical aligned amino acids, i.e., 471 or 472/589*100%=80%. 
     An impaired resistance conferring gene according to the present invention is meant to indicate a gene providing a reduced, or even absent, susceptibility to  Podosphaera leucotricha  as indicated by powder-like spots on the leaves and stems. 
     Impaired resistance conferring genes according to the present invention are mutated genes. The mutation, or mutations, in the present genes can results/result in impairment by different mechanisms. For example, one or more mutations in protein encoding DNA sequences can result in mutated, truncated or non-functional proteins. One or more mutations in non-coding DNA sequences can cause alternative splicing, translation or protein trafficking. Alternatively, one or more mutations resulting in an altered transcriptional activity of a gene, which determines the amount of mRNA available for translation to protein, can result in a resistance due to a low level, or complete absence, of encoded proteins. Additionally, the impairment of the present genes may be caused after translation, i.e. at protein level. 
     Impaired is also indicated herein as encoding a non-functional gene or protein. Although the function of the present genes is not yet identified, a non-functional gene or protein can be readily determined by establishing  Podosphaera leucotricha  resistance (non-functional) or  Podosphaera leucotricha  susceptibility (functional) in a plant. A  Podosphaera leucotricha  resistance (non-functional) plant is indicated by comprising a gene which is mutated at the protein level as compared to the SEQ ID No. 1 or no or reduced levels are observed of mRNA comprising SEQ ID No. 2. 
     Functional and non-functional genes, or proteins, can also be determined using complementation experiments. For example, transforming a  Podosphaera leucotricha  resistant  Malus domestica  plant with a copy the present genes under the control a constitutive promoter will result in a  Podosphaera leucotricha  susceptible  Malus domestica  plant. 
     According to the present invention, the present  Podosphaera leucotricha  resistance conferring genes provide  Podosphaera leucotricha  resistance when impaired. Impaired according to the present invention can be indicated by the absence, or decrease of a protein identified herein by SEQ ID No. 1. In the art, many mechanisms are known resulting in the impairment of a gene either at the transcription, translation or protein level. 
     For example, impairment at the transcription level can be the result of one or more mutations in transcription regulation sequences, such as promoters, enhancers, initiation, termination or intron splicing sequences. These sequences are generally located 5′ of, 3′ of, or within the coding sequences represented by SEQ ID No. 2. Impairment can also be provided by a deletion of, rearrangement of or insertion in the present genes. 
     Impairment at the translation level can be provided by a premature stop-codons or other RNA to protein controlling mechanisms or posttranslational modifications influencing, for example, protein folding or cellular trafficking. 
     Impairment at the protein level can be provided by truncated, misfolded or disturbed protein-protein interactions. 
     Independent of the underlying mechanism, impairment according to the present invention is indicated by a decrease, or absence, a functional protein according to SEQ ID No. 1. 
     Considering the above, according to an embodiment of the first aspect of the present invention, impairment according to the present invention comprises one or more mutations in the present genes resulting in the absence of a protein expression product with a primary amino acid sequence represented by SEQ ID No. 1 or an mRNA comprising SEQ ID No. 2. 
     According to another embodiment of this first aspect of the present invention, the present impairment comprises one or more mutations in the present genes resulting in a non-functional protein expression product. 
     According to still another embodiment of this first aspect of the present invention, the present impairment comprises a reduced transcription level resulting in a reduced level of an mRNA comprising SEQ ID No. 2. 
     According to yet another embodiment of this first aspect of the present invention, the present impairment comprises a reduced translation level of an mRNA comprising SEQ ID No. 2. 
     According to an especially preferred embodiment of the invention, the present  Podosphaera leucotricha  resistance conferring gene is derived from  Malus domestica.    
     According to a second aspect, the present invention relates to  Malus domestica  plants comprising in their genome an impaired  Podosphaera leucotricha  resistance conferring gene as described above wherein the impairment provides  Podosphaera leucotricha  resistance. 
     According to a preferred embodiment of this second aspect of the present invention, the present  Malus domestica  plants show an expression, or transcription, of the present  Podosphaera leucotricha  resistance conferring genes being reduced by at least 10% as compared to a  Malus domestica  plant susceptible to  Podosphaera leucotricha , preferably wherein the expression, or transcription is reduced by at least 20% as compared to a  Malus domestica  plant susceptible to  Podosphaera leucotricha , preferably at least 30%, more preferably at least 50%, even more preferably at least 70%, and most preferably at least 80% such as 25%, 35%, 40%, 45%, 55%, 60%, 65% or 75%. 
     According to another preferred embodiment of this second aspect of the present invention, the present  Malus domestica  plants display an absent expression, or transcription of the present  Podosphaera leucotricha  resistance conferring genes. 
     According to an especially preferred embodiment of this second aspect of the present invention, the present  Malus domestica  plants comprise in their genome an impaired  Podosphaera leucotricha  resistance conferring gene encoding a protein with the primary amino acid sequence of SEQ ID No. 1, or a primary amino acid sequence with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity with SEQ ID No. 1; Formulated differently, the present invention relates according to an especially preferred embodiment to  Malus domestica  plants comprising an impaired Mdmlo19 gene or comprising an impaired MdMLO19 gene. 
     According to a third aspect, the present invention relates to seeds, plant parts or propagation material of the present  Podosphaera leucotricha  resistant plants comprising in their genome the present an impaired  Podosphaera leucotricha  resistance conferring gene providing  Podosphaera leucotricha  resistance. 
     According to a fourth aspect, the present invention relates to isolated nucleotide sequence represented by SEQ ID No. 2, or nucleotide sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity therewith. 
     According to a fifth aspect, the present invention relates to isolated amino acid sequences represented by SEQ ID No. 1, or amino acid sequences with more than 70% identity, preferably more than 80% identity, more preferably more than 90% identity, and most preferably more than 95% identity therewith. 
     According to a sixth aspect, the present invention relates to the use of the present  Podosphaera leucotricha  resistance conferring gene, the present isolated nucleotide sequence or the present isolated amino acid sequence for selecting an  Podosphaera leucotricha  resistant  Malus domestica  plants using, for example, the present sequence for developing molecular markers. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The present invention will be further detailed in the example below. In the example, reference is made to figures wherein: 
         FIG. 1 : shows the area under disease progress curve (AUDPC) of four apple mlo lines and of the control ‘Gala’, inoculated with  Podosphaera leucotricha . Average AUDPC was calculated from 15 to 24 biological replicates considered in four experiments. Error bars show the standard errors of the mean. Statistically significant differences in the comparisons with ‘Gala’, according to Tukey and Games-Howell post-hoc tests (P=0.05) are indicated with asterisks. 
         FIG. 2 : shows the number of conidia per cm 2 leaf surface of ‘Gala’ and mlo lines TG0, TG11, TG19 and TG11+19 inoculated with  P. leucotricha  at 21 dpi. Bars indicate the average number of conidia, measured in two experiments. Error bars show standard errors of the mean. Asterisks indicate statistically significant differences compared to ‘Gala’ according to Tukey post-hoc test (P=0.01). 
         FIG. 3 : shows bright field microscopy images of infected leaves of ‘Gala’ and lines TG11+19 and TG19 taken at 3, 10, and 21 dpi. For Gala at 3 dpi, at higher magnification the germination of a  P. leucotricha  spore is shown. 
         FIG. 4 : shows SEM microscopy images of infected leaves of ‘Gala’, the susceptible line TG0 and the resistant line TG11+19. Pictures were taken at 21 dpi. 
         FIG. 5 : shows formation at 3 dpi of papillae in infected leaves of ‘Gala’ (A, B) and in resistant lines TG11+19 (C, D) and TG19 (E). Images on the left were taken with a bright field microscope, those on the right with fluorescence microscope. For line TG19 only the image taken with the fluorescent microscope is shown. 
         FIG. 6 : shows expression of two apple MLO genes in five mlo lines in absence of  P. leucotricha  infection. Each bar represents the line average relative expression, evaluated from three to five plants. Error bars show the standard errors of the mean. Asterisks indicate significant differences in the comparison of mlo lines with ‘Gala’, based on Tukey or Games-Howell post-hoc tests (P=0.05). 
     
    
    
     DESCRIPTION OF THE INVENTION 
     EXAMPLE 
       Malus  is a genus composed of numerous widely dispersed and diverse taxa, yet nearly all of the world&#39;s commercial apple production is concentrated within a single species,  Malus domestica , which is native to Asia.  M. domestica  is highly susceptible to PM, with relatively minor differences among cultivars and selections. 
     The control of PM in commercial orchards generally requires the significant use of fungicides, which has an economical impact for apple producers. Numerous organic and inorganic fungicides are used to control  P. leucotricha . However  P. leucotricha  has exhibited hability to develop resistance to these materials when they are used repeatedly, and also these synthetic products are not environmentally friendly. 
     To improve the resistance of apple to this disease, breeders began to introduce genetic resistance from wild  Malus  spp. into  M. domestica , resulting in many  Malus  hybrids (such as White Angel). 
     Three binary RNAi vectors were developed to silence 2 MLO genes. Inoculation of transgenic “Gala” mutated for the MLO genes, showed that silencing of the MdMLO19 gene increase the resistance of  M. domestica  to  P. leucotricha . A decrease up to 75% of the susceptibility to this disease was observed in the mutated “Gala” line with a level of silencing of MLO around 90%. 
     Screening the resequencing data of 63 apple cultivars, we discovered a natural loss-of-function mutation in MdMLO19. No similar mutations were found in the other four MLO genes considered (MdMLO5, 7, 11 and 18). When this mutation is present at both alleles, the apple cultivar is resistant. 
     Materials and Methods 
     Constructs for MdMLO11 and MdMLO19 Knock-Down in Apple 
     Gene fragments for RNAi were amplified from MdMLO11 and MdMLO19 (accession numbers in Table 1) with primers listed in Table 2 and cloned in gateway pENTR/SD-TOPO (Thermo Fisher Scientific, Waltham, USA). In addition, a chimeric construct was developed joining RNAi fragments supposed to silence MdMLO11 and MdMLO19 simultaneously (Abbott et al., 2002). For this purpose, a restriction site for EcoRI was added at the 3′ end of the MdMLO11 RNAi fragment and at the 5′ end of the MdMLO19 one. Both fragments were restricted with EcoR1 and joined with a T4 DNA ligase (New England Biolabs, Ipswich, USA). The resulting construct was cloned into the pENTR vector. After sequencing, all fragments were cloned into the destination vector pHELLSGATE 12 (Thermo Fisher Scientific, Waltham, USA). The final constructs were verified by sequencing, and inserted into  A. tumefaciens  strain AGL0 through electroporation.  A. tumefaciens  transformed cells were tested by PCR for the presence of the constructs, using specific primers designed to anneal on vector and MLO sequences. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Primers for qPCR 
               
            
           
           
               
               
               
               
            
               
                   
                 Accession 
                   
                   
               
               
                 Name 
                 number 
                 Forward (′5-′3) 
                 Reverse (′5-′3) 
               
               
                   
               
               
                 EF1 
                 MD09G014760 a   
                 TACTGGAACATCACAGGCTGAC 
                 TGGACCTCTCAATCATGTTGTC 
               
               
                   
                   
                 (SEQ ID NO: 3) 
                 (SEQ ID NO: 4) 
               
               
                   
               
               
                 Ubiquitin 
                 MD05G001920 a   
                 CATCCCCCCAGACCAGCAGA 
                 ACCACGGAGACGAAGCACCAA 
               
               
                   
                   
                 (SEQ ID NO: 5) 
                 (SEQ ID NO: 6) 
               
               
                   
               
               
                 Md8283 
                 MDP0000375455 b   
                 CTCGTCGTCTTGTTCCCTGA 
                 GCCTAAGGACAGGTGGTCTATG 
               
               
                   
                   
                 (SEQ ID NO: 7) 
                 (SEQ ID NO: 8) 
               
               
                   
               
               
                 MdMLO11 
                 MDP0000239643 b   
                 ATCGAAGGCTGTTGGAGCAA 
                 AAGCACGTGAAAGACGGCTA 
               
               
                   
                   
                 (SEQ ID NO: 9) 
                 (SEQ ID NO: 10) 
               
               
                   
               
               
                 MdMLO19 
                 MDP0000168714 b   
                 CAGAGTGGCGACTGCACTTA 
                 GGGACATGGAGTGCAAAGGA 
               
               
                   
                   
                 (SEQ ID NO: 11) 
                 (SEQ ID NO: 12) 
               
               
                   
               
               
                   a Available at http://bioinformatics.psb.ugent.be/plaza 
               
               
                   b Available at http://www.rosaceae.org/gb/gbrowse/malus_x_domestica/ 
               
            
           
         
       
     
                     TABLE 2                  Primers for RNAi                                     Accession           Amplicon       Gene   number a     Primer Forward   Primer Reverse   Lenght               MdMLO11   MDP0000239643   GCACATCGCAGCGAAGAAGCAC   AGCTTTCAGTGTCCTGTTCGGATTG   134 bp               (SEQ ID NO: 13)   (SEQ ID NO: 14)                   MdMLO19   MDP0000168714   TGCACTTGCTTTCTTTGCATGGAC   AACGACATCTTCCAACTTCTCATGG   115 bp               (SEQ ID NO: 15)   (SEQ ID NO: 16)                 a Available at http://www.rosaceae.org/gb/gbrowse/malus_x_domestica/.            
Development of RNAi Apple Plantlets
 
     The RNAi-constructs were transferred into apple as described by Joshi et al. (2011). Explants from the top four leaves of 4-week-old in vitro propagated shoots of the cultivar Gala were kept on a medium with kanamycin (Joshi et al., 2011), and grown in a growth chamber with 16-h-light/8-h-dark cycle at 24° C. To certify the presence of the constructs with PCR, genomic DNA from regenerated plantlets was extracted with the Illustra Nucleon Phytopure kit (GE Healthcare). The forward primer annealed on the CaMV 35S promoter (5′-CGCACAATCCCACTATCCTT-3′) (SEQ ID NO: 17) and the reverse primers were specific for the RNAi fragments (Table 2). PCR was performed with GoTaq® Green Master Mix (Promega, Fitchburg, USA). Plants positive for the construct were moved to Shoot Propagation Medium (SPM): 4.4 g/L of Murashige and Skoog medium with vitamins, 30 g/L of sucrose, 0.7 mg/L of BAP, 96 mg/L of FeEDDHA, pH 5.8. To promote rooting, plants were transferred on a medium containing IBA to promote rooting. Once roots were formed, plants were progressively acclimated to greenhouse conditions (25° C., 16-h-light/8-h-dark cycle, relative humidity 70±5%) in 125 ml pots covered with plastic bags and containing wet autoclaved turf (“Terriccio Vegetal Radic”—Tercomposti Spa, Brescia, Italy). Every 5-7 days for three weeks, air humidity was reduced to promote the formation of the foliar cuticle. Plastic bags were then removed and plants were transferred to 1 L pots. The control (untransformed in vitro grown ‘Gala’) was acclimated as described above. 
       P. leucotricha  Inoculation and Disease Severity Assessment in Apple 
     To produce a PM inoculum, local strains of  Podosphaera leucotricha  were isolated from infected leaves of an orchard located in the Trentino province (Italy). The fungus was maintained by serial inoculations on  M. domestica  seedlings under greenhouse conditions. Plants were dry-inoculated by brushing the adaxial epidermis with leaves of infected seedlings. To promote the fungal penetration, plants were incubated in greenhouse at 25° C. with a relative humidity of 100% for 6 h. The plants were then maintained at 25° C. and 80±10% relative humidity until the end of the evaluation. 
     Four inoculation experiments were carried out in different periods of the year. In each test, three to eight biological replicates of each transgenic line were considered. Lines were tested in at least three out of four experiments and the total number of replicates varied between 15 and 24. Disease severity was visually assessed on all inoculated leaves 7, 14 and 21 dpi. Disease severity was expressed as the percentage (intervals of 5%) of adaxial leaf area covered by the PM mycelium, and a single plant mean value was calculated. Reduction of disease severity in transformed plants was expressed as [(severity in controls−severity in transgenics)/severity in controls]×100%. To consider all time points together, the area under the disease progress curve (AUDPC), summarizing disease intensity over time (Campbell and Madden, 1990; Madden et al., 2007), was calculated. The number of  P. leucotricha  conidia present on infected leaves was assessed as in Angeli et al. (2012) with slight modifications: three leaves were collected from each replicate at 21 dpi and four disks of 0.8 cm diameter for each leaf were cut for a total of 12 per replicate. Leaf disks were transferred to 50 ml tubes containing 5 ml distilled water with 0.01% Tween 20 (Sigma-Aldrich, Saint Louis, USA). Tubes were vortexed for one minute and the concentration of conidia per ml was determined by counting with a hemocytometer under a light microscope. The amount of conidia was expressed as number per square centimeter (cm 2 ) of leaf. 
     Histological Analysis of Inoculated Apple Leaves 
     Two inoculated leaves for each replicate were collected at 3, 10 and 21 days post inoculation for bright field microscopy observations. To visualize fungal hyphae, leaves were cleared in ethanol:acetic acid (3:1 v/v) until chlorophyll removal (approximately 48 hours). Samples were stained for 15 minutes with 250 μg/ml trypan blue in lactic acid, glycerol, and water (1:1:1). After rinsing and mounting as in Vogel and Somerville (2000), hyphae were visualized under bright field illumination of a Leica LMD7000 microscope (Wetzlar, Germany). 
     Leaves considered for scansion electron microscopy (Hitachi S-2300, Tokyo, Japan) were fixed in Sorensen phospate buffer 0.1 M, pH 7, 3% gluteraldehyde. After 24 hours, leaves were washed in Sorensen buffer without gluteraldehyde for two hours under mild agitation (80-100 rpm). Afterwards, samples were progressively dehydrated with four ethanol washings at concentrations from 40 to 100%, dried and kept in falcon tubes until observation. Fragment of leaves were metallized with gold before observation. Images were processed with ImageJ software (http://imagej.nih.gov/ij/). 
     For the detection of papillae, leaves were cleared in ethanol:acetic acid (3:1, v/v) until chlorophyll removal, and equilibrated overnight in a solution of lactic acid, glycerol and water (1:1:1). Papillae were visualized using the LMD filter (BP filter 380-420 nm excitation, 415 dichroic mirror, and BP 445-485 nm emission) of a Leica LMD6500 microscope (Leica Microsystem, Wetzlar, Germany). 
     Gene Expression Analysis 
     To identify lines showing silencing effects, a first gene expression study used in vitro grown transgenic plants replicated three times. In the second study concerning acclimated transgenic plants, leaf samples were collected immediately before PM inoculation, at 24 hpi and at 10 dpi. For each line at each time point, leaf samples were collected from five different plants. Samples were frozen in liquid nitrogen and stored at 80° C. Total RNA was extracted with the Spectrum™ Plant Total RNA kit (Sigma-Aldrich), treated with the DNAse I (Sigma-Aldrich) and reverse transcribed using the SuperScript III reverse transcriptase (Invitrogen, Life Technologies, Waltham, USA). The qPCR analyses were run according to SsoAdvanced Universal SYBR Green Supermix, (Bio-Rad, Hercules, USA) in a 15-μ1 reaction volume, using a CFX96 Touch™ Real-Time PCR detection system (Bio-Rad, Hercules, USA), and the CFX Manager software. Samples were run in two technical replicates according the following thermal cycling parameters: 95° C. 3 min, 95° C. 10 sec, 55° C. 30 sec (repeated 40 times), 95° C. 10 sec. For the analysis of MdMLO19, the primer pairs considered in previous work were used (Table 1; Pessina et al., 2014). For MdMLO11 and for the expression of 17 genes involved in the interaction between apple and  P. leucotricha , new primer pairs were designed with the NCBI Primer Designing Tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table 1). Serial dilutions of cDNA (1/10, 1/100, 1/1000 and 1/10000) allowed to calculate the efficiency of the primer pairs; the expected sizes of the products were confirmed using agarose gel electrophoresis. Presence of a specific final dissociation curve was determined after every qPCR run, with progressive increments of temperature from 65° C. to 95° C. (0.5° C. each step, 5 sec). The reference genes considered were elongation factor 1, ubiquitin and 8283 (Table 1). All of them are known to be stable reference genes for apple (Botton et al., 2011; Pessina et al., 2014). The analysis with the software geNorm (medgen.ugent.be/˜jvdesomp/genorm) resulted in M-values lower than 1 for all three reference genes, in conditions where M-values lower than 1.5 are considered adequate (Ling and Salvaterra, 2011). The threshold cycles (Ct) were converted to relative expression levels as in Hellemans et al. (2007), using as input the average Ct of the two technical replicates. As reference Ct, the average Ct of wild-type ‘Gala’ at 0 hpi was adopted. 
     Statistics 
     Disease Severity 
     Severity data were analysed by the statistical package SPSS (IBM, Armonk, USA). For both apple and  A. thaliana , severity data of leaves from the same plant were averaged before further analyses. Apple severity data of the eight younger leaves of a plant were considered, while  A. thaliana  data were from all leaves. Before any analysis, data were shown to be normally distributed (Kolmogorov-Smirnov and Shapiro-Wilk tests P&gt;0.05) and to have homogeneous variances (Levene&#39;s test, P&gt;0.05). One-way ANOVA with Tukey&#39;s post-hoc test was adopted to detect significant differences (P&lt;0.05) at each time point. Data were transformed according to y=arcsin(x), in order to meet the pre-requisites of ANOVA. In case of non-homogeneous variances, the Games-Howell&#39;s post-hoc test was applied. Prior to pooling data from independent experiments, the effect of single experiments was tested: no significant effect of the experiments emerged. Pooled data were analysed independently for time points 14 and 21 dpi. AUDPC data were treated as described above for severity data. Number of conidia data was analysed with one-way ANOVA, applying the Tukey post-hoc test (P&lt;0.05). 
     qPCR Data Analyses 
     For the evaluation of gene expression, relative expression values were transformed in logarithmic scale according to Y=ln(x) (Pessina et al., 2014) to meet normal distributions and homogeneities of variances, as assessed respectively with the test of Shapiro-Wilk (P≤0.05) and Levene (P≤0.05). Pairwise comparison of homoscedastic data was carried out with Tukey&#39;s test (P&lt;0.05), whereas non-homoscedastic data were analysed with Games-Howell test (P&lt;0.05), using the statistical package SPSS (IBM). To detect significant differences in expression, one-way ANOVA with Tukey post-hoc test (P&lt;0.05) was applied to data from samples collected at 0 hpi. Defense gene expression analysis, was tested with the Fisher post-hoc test. 
     Correlations 
     The two-tailed Pearson&#39;s correlation test was adopted to investigate the correlations between AUDPC and relative expression of MLO genes at 10 dpi, and between degree of severity and number of conidia, both at 21 dpi. All data have been transformed following y=arcsin(x) to achieve a normal distribution. 
     Metabolites 
     The data from the phenolic metabolites were subjected to one-way ANOVA with Fisher post-hoc test. In case of non-homoscedastic data, the Games-Howell post-hoc test was applied, and the Kruskall-Wallis non-parametric test for data not normally distributed. 
     Results 
     Development of RNAi Apple Plantlets 
     Three RNAi constructs were generated, two aimed at knocking-down MdMLO11 and MdMLO19 individually (i=KD-MdMLO11, ii=KD-MdMLO19), the third aimed at the simultaneous knock-down of MdMLO11 and MdMLO19 (iii=KD-MdMLO11+19). Eighty regenerated lines were obtained of which 48 did carry the RNAi insert as described in materials and methods (Table 3). The 48 transgenic lines were tested by qPCR to evaluate the level of MLO genes expression, but a significant knock-down was observed only in three of them (Table 3). In these three lines, off-target knock-downs were not detected for the other two clade V genes of apple (MdMLO5 and 7). The three Knock-down lines, named TG11 (Transgenic Gala MdMLO11), TG19 and TG11+19, were acclimated to greenhouse conditions, as well as the control wild-type ‘Gala’ and TG0, a line carrying the RNAi construct for MdMLO19 but not showing significant MLO genes knock-down. TG0, TG11, TG19 and TG11+19 will be indicated as transgenic lines, but only TG11, TG19 and TG11+19 as mlo lines. 
     The survival rate of plants to the acclimation procedure was above 90%. Under greenhouse conditions the mlo lines showed a normal growth compared to ‘Gala’ under greenhouse conditions. 
                     TABLE 3                  summary of gene transfer results                                 Gene       Regenerated   Confirmed   Selected       transfer   Knocked-down gene   lines   transgenic   lines                                         i   MdMLO11   39   23   TG11       ii   MdMLO19   33   19   TG0, TG19       iii   MdMLO11 +   8   5   TG11 + 19           MdMLO19                    
Reduced Susceptibility to  P. leucotricha  of RNAi Apple Plants
 
     The four transgenic lines and the control were tested for their susceptibility to PM in four independent experiments. TG0, the line not manifesting any MLO genes knock-down, showed a level of susceptibility to  P. leucotricha  comparable to that of the control; the same was noted for TG11, whereas TG11+19 and TG19 had an evident reduction of disease severity ( FIG. 1 ). Although leaves of TG11+19 and TG19 plants were partially infected ( FIG. 1 ), the extension of the adaxial leaf area covered in spores was significantly reduced compared to the control ( FIG. 1 ). Table 4 summarizes the results on disease severity reduction. 
     All the transgenic lines had a reduction in the number of conidia present on leaves ( FIG. 2 ), but the decrease was statistically significant (P&lt;0.05) only for TG11+19 and TG19. This compares well with the disease severity assessment presented in  FIG. 1 : compared to ‘Gala’, TG11+19 showed a 63.3% reduction in the number of conidia, and TG19 of 64.8%. A significant (P=0.01) but moderate positive correlation (Pearson coefficient of 0.525) was found between disease severity at 21 dpi and conidia count at 21 dpi. 
     Lines TG11+19 and TG19, together with ‘Gala’, were further analysed by bright field microscopy and scansion electron microscopy (SEM), to follow the development of  P. leucotricha  infection. In ‘Gala’, a well-developed leaf infection was observed already at 3 dpi (FIG.  3 A), at the time when fungal development was still limited in TG11+19 and TG19 ( FIGS. 3B and 3C ). At 10 dpi, conidiophores were observed on leaves of all lines considered, but their number was higher in ‘Gala’ ( FIG. 3 ). At 21 dpi, ‘Gala’ leaves were completely covered by spores and a large number of conidiophores were visible ( FIG. 3A ). The leaf surface of TG11+19 and TG19 was partially colonized by sporulating mycelium, but isolated spores unable to develop were also observed, as well as a smaller number of conidiophores compared to the situation noted for ‘Gala’ ( FIGS. 3B and 3C ). The SEM images showed reduced growth of the mycelium on TG11+19 compared to TG0 and ‘Gala’ ( FIG. 4 ). 
     The formation of papilla was observed at 3 dpi in all the lines, both resistant and susceptible ( FIG. 6 ). Compared to TG11+19 and TG19, the papillae of ‘Gala’ ( FIG. 6A ,B) were smaller, the shape more defined and the fluorescence emitted was more intense ( FIG. 6C ,D,E). 
                     TABLE 4                  Powdery mildew disease severity reduction (%)       in lines transformed with MLO RNAi constructs.                                 Number                   of repli-   Disease severity reduction #                                           Silenced genes   cates   14 dpi   21 dpi   Average                                                 TG0   /   17   24.1   24.8   24.5       TG11 +   MdMLO11   23   60.0*   52.6*   56.3       19   and 19       TG19   MdMLO19   15   72.7*   78.1*   75.4       TG11   MdMLO11   16   38.0   −3.2°   17.4               *Statistically significant difference compared to the control, according to the Tukey post-hoc test (P = 0.05).         # Gala was used as control (19 plants) and assumed to have 0% of disease reduction.       °Line TG11 showed a higher level of infection compared to Gala at 21 dpi.            
Expression of MLO Genes in mlo Apple Lines
 
     Gene expression analysis of mlo lines previously selected was repeated in greenhouse acclimated plants. MdMLO11 was significantly less expressed in TG11+19 (P=0.01) and TG11 (P=0.05) ( FIG. 6A ), whereas the expression of MdMLO19 was reduced in TG11+19 (P=0.01) and TG19 (P=0.01) ( FIG. 6B ). MdMLO5 and MdMLO7, the two other apple members of Clade V, were also tested but no significant reduction was observed in any transgenic line, a finding supporting the absence of off-target silencing (data not shown). 
     Correlation between the expression of MdMLO19 and AUDPC—a measure of disease severity—was statistically significant (P=0.05), although moderate (Pearson coefficient=0.515). On the contrary, no significant correlation was found between AUDPC and the expression of MdMLO11.
 
Discussion
 
     Natural and artificial loss-of-function mutations of MLO S-genes reduce susceptibility to PM pathogens, as described in barley (Biischges et al., 1997),  A. thaliana  (Consonni et al., 2006), pea (Pavan et al., 2011), tomato (Bai et al., 2008) and pepper (Zheng et al., 2013). In dicots, all PM-susceptibility genes belong to Clade V (Consonni et al., 2006; Bai et al., 2008; Feechan et al., 2008; Winterhagen et al., 2008). In a previous contribution we identified three MLO genes of  M. domestica  up-regulated during early stages of PM infection (Pessina et al., 2014). Two of them, MdMLO11 and MdMLO19, belong to dicot clade V and MdMLO18 to clade VII. Because MLO genes outside clade V acting as S-genes are not known, only MdMLO11 and MdMLO19 were considered reasonable candidates to be knocked-down in apple. 
     MdMLO11 and MdMLO19 were knocked-down to assess their role in supporting apple susceptibility to PM. RNAi was adopted to reduce the expression of the two MLO genes, and in spite of the high number of transgenic ‘Gala’ lines generated (48), only for three of them a significant reduction of expression of the target genes was detected. In part, this was expected because short RNAi fragments of less than 150 bp, like those used in our experiments, are known for their limited knock-down efficiency. On the other hand, they have the advantage of being more specific, thus avoiding the generation of off-target silencing of other clade V MLO genes, as detected in our experiments. 
     In some species, knock-out or knock-down of MLO genes causes pleiotropic phenotypes, such as formation of necrotic spots on leaves and reduced grain yield in barley (Jorgensen, 1992), slow growth in  A. thaliana  (Consonni et al., 2006) and reduced plant size in pepper (Zheng et al., 2013). Such or other unexpected pleiotropic phenotypes were not observed under the greenhouse conditions specified in Materials and Methods. 
     Greenhouse inoculation of apple transgenic lines resulted in a statistically significant reduction of disease severity in lines TG11+19 and TG19. Because of the knock-down of MdMLO19 in both resistant lines, it was assumed that this was the most effective gene responsible for the reduction of PM susceptibility. The knock-down of MdMLO11 did not result in a significant reduction of susceptibility and even its knock-down in combination with MdMLO19 resulted in any additional reduction of susceptibility. The conclusion is that out of the two Clade V genes induced by PM in apple, only MdMLO19 is a functional S-gene. Also MdMLO18, the Clade VII gene inducible by  P. Leucotricha  inoculation, should not be considered a PM S-gene. Line TG0 was considered with the purpose of assessing the effect on susceptibility to PM of the insertion of a “target ineffective” RNAi construct. TG0 was obtained from a transfer that aimed to knock-down MdMLO19. In the line, a decrease of expression of MdMLO19 was recorded, although not significant, as well as a moderate non-significant reduction of PM susceptibility. It is concluded that the insertion of an “ineffective” RNAi construct may have functional relevance, but this cannot be statistically proved. 
     The precise mechanism through which the loss-of-function of MLO S-genes reduces susceptibility to PM pathogens is not completely clear yet. However, mlo resistance is known to be linked to secretory vesicles traffic (Miklis et al., 2007; Feechan et al., 2011) and to the formation of cell wall appositions called papillae (Consonni et al., 2006). Papillae consist in a callose matrix enriched in proteins and autofluorogenic phenolics (Vanacker et al. 2000) whose formation depends on actin-dependent endomembrane transport (Hückelhoven, 2014). Lines ‘Gala’, TG11+19 and TG19 were characterized by the presence of papillae at 3 dpi, but shape and dimensions were different in resistant and susceptible lines. Rapid papilla formation (Lyngk r et al., 2000), increased papilla size at attempted penetration sites (Stolzenburg et al., 1984) and different biochemical composition (Chowdhury et al., 2014), may explain the noted differences between effective and non-effective papillae. In mlo lines, particularly in TG19, the size of papillae was larger than in the control, supporting the hypothesis that larger dimensions increase the efficacy of the papilla. Chowdhury et al. (2014) have shown that the difference between effective and non-effective papillae lies in the higher concentration of callose, cellulose and arabinoxylan of the effective ones. This possibly reflects the observed differences in fluorescence between papillae of resistant and susceptible lines. As a matter of fact, MLO proteins are considered negative regulators of vesicle-associated and actin-dependent defense pathways (Panstruga, 2005), which, once under the control of the fungus, induce actin filaments to supply nutrients for the growing hyphae (Miklis et al., 2007). The data presented here support the view that in apple wild-types, after penetration the pathogen controls the transport of material to the cell-wall, changing the composition of the papillae and turning them into non-effective. A similar work carried out in grapevine (Pessina et al., unpublished) support this interpretation: compared to the control, mlo grapevine lines showed larger and less defined papillae, similar to those observed in mlo apple. 
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