Nectar- and stigma-specific expression of a chitinase could partially protect against fire blight in certain apples

To attract pollinators many angiosperms secrete stigma exudate and nectar in their flowers. As these nutritious fluids are ideal infection points for pathogens, both secretions contain various antimicrobial compounds. Erwinia amylovora, the causing bacterium of the devastating fire blight apple disease, is the model pathogen that multiplies in flower secretions and infects through the nectaries. Although Erwinia resistant apples are not available, certain cultivars are tolerant. It was reported that in stigma infection assay, the ‘Freedom’ cultivar was Erwinia tolerant while the ‘Jonagold’ was susceptible. We hypothesized that differences in the nectar protein compositions lead to different susceptibility. Indeed we found that an acidic chitinase III protein (Machi3-1) selectively accumulates in the nectar and stigma of the ‘Freedom’ cultivar. We demonstrate that MYB binding site containing repeats of the ‘Freedom’ Machi3-1 promoter are responsible for the strong nectar- and stigma-specific expression. As we found that in vitro the Machi3-1 protein impairs growth and biofilm formation of Erwinia at physiological concentration, we propose that the Machi3-1 contribute to the tolerance by inhibiting Erwinia multiplication in the stigma exudate and in the nectar. We show that the Machi3-1 allele was introgressed from Malus floribunda 821 into different apple cultivars including the ‘Freedom’. Highlight Certain apple cultivars accumulate to high levels in their nectar and stigma an acidic chitinase III protein that can protect against pathogens including fire blight disease causing Erwinia amylovora


Stain-free protein profiles and western-blot assays 27
Nectars and the protein extracts were separated by stain-free 1D SDS-PAGE (Bio-Rad's Mini 28 PROTEAN® TGX Stain-Free™ Gels). For western blot assays, samples were separated by 29 SDS-PAGE, blotted onto Amersham Protran membrane (GE Healthcare, 10600008) and 30 hybridized with rabbit polyclonal antibody serum raised against Machi3-1. ECL Anti-Rabbit 31 IgG Horseradish Peroxidase linked (GE Healthcare, NA934-1ML) secondary antibody was 32 used for detection. Actin antibody (Anti-Actin Plant MerckA0480) was used for control. Chemiluminescent protein detections were conducted with ECL Western Blotting Substrate 1 (Promega, W1001), according to the manufacturer's instructions. Western blots were scanned 2 with ChemiDoc MP System and analyzed with ImageLab 5.0 software (Bio-Rad). 3 4 Protein sequencing 5 The dominant protein band of 'Freedom' nectar was partially sequenced (described in details 6 at Supplementary Materials and Methods S1). Briefly, the excised protein band was in-gel 7 digested as described (Migh et al., 2018). Peptides were analyzed by data-dependent LC-MS 8 using a Waters Q-TOF Premier mass spectrometer online coupled to a nanoAcquity uHPLC 9 system. Raw data was converted into a peaklist using the ProteinLynx PLGS software and the 10 data was searched using the Batchtag Web software of the Protein Prospector search engine. 11 As automated protein identification did not yield high confidence identifications, MS/MS data 12 were inspected manually and high-quality MS/MS spectra were evaluated manually. Protein 13 segments were used for degenerate PCR primer designing. 14 15 PCR cloning of the Machi3-1 gene from Freedom cultivar 16 Degenerated oligonucleotides (aldegf and aldegr, respectively) were designed for the 17 predicted N-proximal ADYIWNNF and the C-proximal WNRFYDN peptide segments. 18 cDNA was prepared from total RNA isolated from the nectary rich tissues of 'Freedom'. PCR 19 product was amplified, subsequently cloned and sequenced. Based on this information 20 specific oligonucleotides were synthesized to clone the genomic region by inverse PCR (invj1 21 for, invj2 for, invba1 rev, invba2 rev). 22

RT-PCR assays 24
For quantitative RT-PCR, total RNAs were treated with DNase I (Thermo Fisher Scientific, 25 EN0525), and cDNAs were transcribed using a RevertAid First Strand cDNA Synthesis kit 26 (Thermo Fisher Scientific, K1621). qRT-PCR was carried out with Fast Start Essential DNA 27 Green Master Mix (Roche, 06402712001) in a Light Cycler 96 Real-Time PCR instrument 28 (Roche). For semi-quantitative RT-PCR assays, the same cDNAs were used in conventional 29 The promoter regions of Machi3-1 alleles were amplified with the SaFreeFor and SaFreeRev 1 primers and separated on 1.5 % agarose gel. 2 3

Plant transformation 15
Leaf disc transformation was carried out to generate transgenic N. tabaccum plants (Bevan et 16 al., 1985). Transgenic tobaccos were selected on kanamycin containing media and then the 17 regenerants (T 0 plants) were grown in the greenhouse. 18

19
Expression of recombinant proteins in Pichia pastoris, analysis of protein expression peptide of Machi3-1 transports the protein to the extracellular space, allowing the purification 23 of the protein from the supernatant. The secreted protein was purified from the supernatant by 24 precipitating with ammonium-sulfate (60% saturation). The precipitate was pelleted (15000 25 rpm, 10 min., 4°C), resuspended in 5 ml 10 mM Sodium-acetate (pH 5.0) and concentrated 26 with Amicon Ultra-4 Centrifugal Filter Units (Merck Millipore, 10K) ~10 fold, reaching the 27 final protein concentration ~3 mg/ml. Empty pPICz vector transformant P. pastoris was 28 grown and induced as the test strain, and then its supernatant was similarly treated 29 (ammonium-sulfate precipitated, centrifuged, resuspended in 10 mM Sodium-acetate and 30 concentrated to ~3 mg/ml). The purified supernatant of the empty vector transformant P.
that the background of the negative control and the purified Machi3-1 was similar (Fig. S2A  1 and S2B). 2 3 Chitinase and lysozyme activity assay 4 Chitinase activity was measured by Schales' reagent method (Ferrari et al., 2014). Colloid 5 chitin was prepared according to Shen (Shen et al., 2010) with minor modifications. 6 g chitin 6 was suspended in 200 ml 37 % HCl and agitated overnight at 4°C. 1 L of distilled water was 7 added followed by centrifugation at 8000 g for 20 minutes. The pellet was washed with water 8 till the pH reached 5.0. 9 Colloid chitin (at 3 mg/ml final concentration) was incubated in 200 µl of 50 mM KPO 4 (pH 10 6.0.) with increasing amounts (50-400 ng) of Machi3-1 protein.

In vitro Erwinia growth inhibition assay 24
Bacterial in vitro growth inhibition assay was carried out with minor modificiations as 25 described (Nash et al., 2006). Approximately 10 2 E. amylovora cells were suspended in 200 26 µl of 10 mM PBS (pH 7.4). The suspension was incubated without shaking for 24 hours at 27 28°C with different amount of purified Machi3-1 protein, or with purified supernatant of 28 empty vector transformant P. pastoris as a negative control. Viable cells were counted by 29 plating. 30 of the diluted culture was incubated at 30°C in a 96-well TC-treated Tissue culture 3 polystyrene plate (1 x 10 6 cells per well) to allow biofilm formation. After 24 hours the 4 suspension was removed, and then 130 µl of purified Machi3-1 diluted in 50mM KPO 4 buffer 5 (pH 6.0) was added to the biofilm covered plates. The reactions were kept at 28°C for 3 hours. 6 Wells were washed 3 times with dH 2 O, then 150 µl 0.1% crystal violet (CV) was added. After 7 15 minutes CV was removed, then the plate was washed 3 times with dH 2 O, and dried for 8 overnight. For quantification 30% acetic acid was added to each well, incubated for 15 9 minutes and the OD was measured at 550 nm. 10 11 Statistics 12 Bacterial growth inhibition assays were repeated four times in independent experiments. 13 Biofilm detachment experiments were performed in octuplicate and repeated three times. 14 Comparisons between groups were done by ANOVA and Tukey-test to determine P-values. 15 Statistical significance * was set at p<0,05 and *** p<0,001. 16 template and the Machi3-1 protein was carried out by the SPDBV (Swiss-PdbViewer) 23 To test our hypothesis that the nectar composition of the fire blight tolerant and susceptible 30 apple cultivars is different, nectar protein profiles of the tolerant 'Freedom', the susceptible proteins accumulated to high levels in the susceptible cultivars, while the nectar of 'Freedom' 1 contained a 29kDa dominant protein (Fig. 1A). Although this protein was present at very high 2 concentration (~50-80 ng/µl) in the 'Freedom' nectar, it was not detectable in the nectars of 3 the susceptible cultivars (Fig. 1A). The analysis was repeated in four consecutive years with 4 the same results, therefore the presence of this dominant protein in the 'Freedom' nectar was 5 not due to any environmental condition. The 29kDa protein was isolated, partially sequenced, 6 then primers were designed and inverse PCRs were conducted to clone the genomic copy of 7 the gene from the 'Freedom' cultivar. The amplified region contained a 894 nucleotide (nt) 8 long intronless coding sequence, a long (1417 nt) upstream and a short (77 nt) downstream 9 regions. Sequence analysis revealed that the predicted 'Freedom' nectar protein is a class III 10 chitinase (will be referred to as Machi3-1 for Malus chitinaseIII-1). Class III chitinases belong 11 to the GH18 endochitinase family (Adrangi and Faramarzi, 2013). Machi3-1 is an acidic class 12 III chitinase (calculated isoelectric point is 4.4), which shows strong sequence similarity 13 (66.4%) to the well characterized class III chitinases as PSC (pomegranate seed chitinase) and 14 Hevamine (64.18%) (Terwisscha Van Scheltinga et al., 1996;Lv et al., 2011;Masuda et al., 15 2015). The critical catalytic amino acids and the cis-peptides (involved in chitin binding) are 16 all conserved. Moreover, homology modelling predicts that the structure of Machi3-1 protein 17 is highly similar to the structure of Hevamine (Fig. S1). Machi3-1 contains an N-terminal 18 signal peptide that destines proteins towards the secretory pathways (Chung and Zeng, 2017). 19 These data suggest that the Machi3-1 protein is a functional, secretable acidic chitinase. 20 21 Machi3-1 is an active chitinase 22 23 Basic class III chitinases frequently have dual chitinase and lysozyme activities, while the 24 acidic class III chitinases have strong chitinase but only weak or no lysozyme activity (Ma et 25 al., 2017). To characterize the Machi3-1 protein, it was expressed in P. pastoris and the 26 chitinase and lysozyme activities of the purified protein were tested in vitro. 27 To measure the chitinase activity of the purified Machi3-1 protein, Schales' procedure using 28 colloidal chitin for a substrate was carried out (Ferrari et al., 2014). S. griseus chitinase and 29 the supernatant of empty vector transformed P. pastoris were used as positive and negative 30 controls, respectively. Machi3-1 proved to be a relatively efficient chitinase; its activity was activity in Micrococcus lysis assays (Fig. S2C). Thus we concluded that Machi3-1, like most 1 acidic chitinase III proteins, has strong chitinase and very weak lysozyme activity. 2 3 4   5 Next we tested if Machi3-1 had an antibacterial effect against E. amylovora. Bacterial cultures 6 were incubated with Machi3-1 protein purified from P. pastoris supernatant or with 7 supernatant of empty vector transformant P. pastoris for a negative control. We found that at 8 high concentration (40-80 ng/µl) Machi3-1 significantly reduced the growth of E. amylovora 9 To analyze the expression pattern of Machi3-1 gene, polyclonal antibody was produced, and 22 accumulation of the Machi3-1 protein was studied in different tissues of the 'Freedom' and 23 'Jonagold' cultivars ( Fig. 3A and Fig. S3). Confirming our earlier data, the Machi3-1 protein 24 accumulated to high levels in the Freedom nectar but was barely detectable in the Jonagold 25 nectar (Fig. S3). Moreover, in the 'Freedom' cultivar the Machi3-1 protein was also abundant 26 in the stigma tissue but accumulated to low levels in the nectary, leaf, petal, stamen and ovary 27 samples. In the 'Jonagold' cultivar, the Machi3-1 protein accumulated to low levels in all 28 samples (Fig. 3A). Next we studied the expression of Machi3-1 at mRNA level in the nectary, 29 stigma and leaf samples of the two cultivars (Fig. 3B). In the 'Freedom' cultivar, Machi3-1 30 transcript expressed to very high levels in both the nectary and the stigma but it was barely 31 detectable in the leaves. By contrast, Machi3-1 mRNA accumulated to low levels in all 32 'Jonagold' samples ( Fig. 3B). Taken into consideration that (i) 5B-Machi3-1 mRNA expressed in the 'Freedom' nectary, while the protein accumulated in the nectar, and that (ii) 1 the Machi3-1 protein has an export signal, we conclude that the 5B-Machi3-1 is a nectarin 2 gene, which expresses in the nectary cells and then its protein product is secreted into the 3 nectar. 4

Machi3-1 inhibits growth and biofilm formation of E. amylovora in vitro
The promoter regions of the 'Freedom' and 'Jonagold' Machi3-1 alleles are different 5 We postulated that variations in the Machi3-1 promoters are responsible for the strikingly 6 different mRNA expression between 'Freedom' and 'Jonagold' cultivars. Therefore, the 7 coding and the promoter regions of the Machi3-1 gene were also cloned from the 'Jonagold' 8 cultivar, and then the 'Freedom' and 'Jonagold' Machi3-1 genes were compared. The 9 nucleotide sequences of the coding regions and the predicted protein sequences are almost 10 identical (only 4/894 nt and 2/298 amino acids are different) indicating that the coding region 11 of Machi3-1 is well conserved in different apple cultivars (Fig. S4). However, while the 12 promoter regions show strong overall similarity, significant differences were found in the 13 middle region of the promoter ( Multiplication of a repeat region in a promoter can dramatically increase transcriptional 29 activity (Espley et al., 2009). We assumed that 5 box containing 5B-Machi3-1 promoter is indirectly for the nectar-and stigma-specific accumulation of Machi3-1 protein in the 1 'Freedom' cultivar. To confirm that the 5B-Machi3-1 promoter is essential for the specific 2 expression, the F1 hybrids from 'Freedom' × 'Red Rome' and 'Freedom' × 'Red Winter' 3 (Free. × R.R. and Free. × R.W.) crosses were studied. The Machi3-1 protein is not detectable 4 in the nectars of the 'Red Rome' and 'Red Winter' cultivars and both are homozygous for the 5 ps-Machi3-1 alleles ( Fig. 4 and Fig. S9). The 'Freedom' harbors one 5B-Machi3-1 and one 6 ps-Machi3-1 allele and contains Machi3-1 protein in the nectar. The F1 progenies segregated 7 close to the 1:1 for 5B-Machi3-1/ps-Machi3-1 heterozygous and for ps-Machi3-1/ps-Machi3-8 1 homozygous plants (Free. × R.R. F1 segregated for 7:7, while Free. × R.W. F1 hybrids 9 segregated for 11:13). Only 6 Free. × R. R. and 8 Free. × R. W. F1 plants developed flower in 10 the year of the study. We found that Machi3-1 protein accumulated to easily detectable levels 11 in the nectar of all F1 progenies that inherited the Freedom 5B-Machi3-1 allele ( Fig. 4 and 12 Fig. S9), while the progenies that inherited the 'Freedom' ps-Machi3-1 allele did not 13 accumulate the protein in their nectar (stigma samples were not collected). These results 14 indicate that the Machi3-1 gene is present in a single copy in 'Freedom', and that the 5B-15 Machi3-1 allele is required and sufficient for the intense nectar-specific (and likely stigma-16 specific) protein expression. 17 The 5B-Machi3-1 promoter can confer nectary-and stigma-specific expression in tobacco 18 If the trans factors that are responsible for the nectar-and stigma-specific expression of the 19 5B-Machi3-1 allele in apple are also present in other dicot plants, the 5B-Machi3-1 promoter 20 can be used as an efficient biotechnology tool for nectary-and stigma-specific expression. To 21 test this assumption transgenic tobacco lines were generated with constructs containing the 22 promoter and the coding region of the 5B-Machi3-1 or the 2B-Machi3-1 alleles (Fig. 5A). We 23 found that the Machi3-1 protein was easily detectable in the nectar of 4 out of 16 5B-Machi3-24 1 transgenic tobacco lines (Fig. 5A). By contrast, the transgenic protein could not be detected 25 in any of the 2B-Machi3-1 transgenic tobacco nectars (0/17 plants). We have also studied the 26 accumulation of the Machi3-1 protein in the stigma of two 5B-Machi3-1 plants that expressed 27 the Machi3-1 protein in their nectars and in two 2B-Machi3-1 plants, which did not 28 accumulate the protein (Fig. 5C and 5D). We found that the expression in the nectar and the 29 stigma correlated. In 5B-Machi3-1 transgenic plants, the Machi3-1 protein accumulated to 30 high levels both in the nectar and the stigma. By contrast, in the 2B-Machi3-1 transgenic 31 plants the Machi3-1 protein could not be detected in either the nectar or the stigma samples. 32 These results show that all trans factors that are required for the specific expression are also 1 present in tobacco. Thus, the 5B-Machi3-1 promoter can be used for efficient nectary-and 2 stigma-specific expression in various dicot plants. 3 The 5 box region of the Machi3-1 promoter is required for efficient expression 4 To directly prove that the boxes of the 5B-Machi3-1 promoter are required for the specific 5 expression, plants were transformed with deletion constructs generated from the 5B-Machi3-1 6 plasmid (Fig. 5B), and then we studied the accumulation of the Machi3-1 protein in the 7 nectars of the transformants (stigma samples were not collected in this experiment). The 8 results suggest that the 5 boxes are essential for the efficient expression, only 1 out of 52 9 plants accumulated Machi3-1 protein in the nectar at detectable levels when constructs 10 lacking the 5 boxes were used (we combined the results of 0.6, 0.4 and 0.2 constructs, see

MYB305 could play an important role in the regulation of 5B-Machi3-1 14
As the 5 box promoter region is required for the efficient and specific expression of the 5B-15 Machi3-1 gene, we assumed that transcription factors that selectively bind to this region play 16 important role in the regulation. To identify 5 box region specific transcription factor binding 17 sites, we compared the 5 box and 2 box promoter regions of the 5B-Machi3-1 and 2Bonly 1 in the 2 box promoter region (Fig. S7). In tobacco, the MYB305 transcription factor 20 directs the nectary-specific expression of many genes including nectarins (Liu et al., 2009). 21 We hypothesized that MYB305 and the apple homolog of MYB305 (referred to as 22 MdMYB305, gene: MDP0000344978) directs the expression of 5B-Machi3-1 in the nectary 23 as well as in the stigma cells. If these assumptions are correct, MYB305 and 5B-Machi3-1 24 (but not 2B-Machi3-1) mRNAs are co-expressed. To test it, qRT-PCR and semi qRT-PCR 25 assays were conducted to monitor the expression of MdMYB305 in 'Freedom' and 'Jonagold' 26 cultivars ( Fig. 6A and Fig. S10A). The MdMYB305 mRNA expressed similarly in both 27 cultivars, it was abundant in the nectary and stigma samples but was barely detectable in 28 leaves ( Fig. 6A and Fig. S10A). As the 'Freedom' 5B-Machi3-1 (but not the 'Jonagold' 2B-Machi3-1 protein was present in the nectar of all the 5B-Machi3-1 allele containing cultivars 23 ( Fig. 7A) but it was not detectable in the nectar of cultivars lacking the 5B-Machi3-1 allele. 24 These data confirm that the 5B-Machi3-1 allele is sufficient for the accumulation of Machi3-1 25 protein in the nectar. 26 Various Vf scab resistant apple cultivars including 'Freedom', 'Releika' and 'Topaz' contain 27 the 5B-Machi3-1 allele (Fig. S11). Although these cultivars were generated in different 28 breeding programs in the USA ('Freedom'), Germany ('Releika') and Czech Republic 29 ('Topaz'), progenies from the M. floribunda 821 clone and 'Rome Beauty' (F 2 26829-2-2) the 5B-Machi3-1 allele was introgressed from the M. floribunda 821 ancestor. Indeed, we 1 found that the M. floribunda 821 contains a 5B-Machi3-1 allele (Fig. 7B), which is almost 2 identical (295/298 amino acids of the predicted proteins are identical) to the Freedom 5Bsimilar to the promoter of the 'Freedom' 5B-Machi3-1 gene, it contains the 5 boxes and all 4 5 MYB binding sites are present (Fig. S12 and S13). We hypothesize that Machi3-1 protein is 6 also abundant in the nectar and the stigma of M. floribunda 821 (flowers were not available to 7 test this prediction). Machi3-1 is present in heterozygous form in M. floribunda 821, the 8 second Machi3-1 allele has a specific promoter with 3 boxes (3B-Machi3-1) (Fig. 7B). Taken  9 together, these data indicate that the 5B-Machi3-1 allele was introgressed from the M. We found that the Machi3-1 acidic chitinase III gene is present in at least three different 23 forms in various apple cultivars, one is a pseudogene, while the two other alleles (5B-24 Machi3-1 and 2B-Machi3-1) encode very similar proteins (Fig. S4). However, the 25 regulation of the two active alleles is markedly different. The 2B-Machi3-1 allele expresses 26 to low levels in all studied tissues, while the 5B-Machi3-1 mRNAs shows very intense 27 expression in the nectary and stigma (Fig. 3). Our segregation, association and transgenic 28 assays demonstrate that the 5 boxes of the 5B-Machi3-1 promoter is required for the 29 nectary-and stigma-specific transcript and for the nectar-and stigma-specific protein MYB21 and MYB24) transcription factor plays a critical role in the expression of nectar 1 proteins in tobacco, snapdragon and Arabidopsis (Roy et al., 2017). MYB305 is activated 2 by JA (and probably by auxin), then it binds to the promoters of many nectary-specific 3 genes (including certain nectarins) and promotes their transcription (MYB305 might also 4 stimulate indirectly the accumulation of other nectar proteins). We identified four potential 5 MYB binding sites on the 5 box region of the 5B-Machi3-1 allele but only one on the 2B 6 promoter (Fig. S7). We demonstrated that in addition to the nectary (Liu et al., 2009), 7 tobacco MYB305 is also expressed to high levels in the stigma (Fig. 6). Moreover, we 8 showed that the apple homolog MdMYB305 transcripts are also abundant in the nectary and 9 stigma tissues, while it accumulates to low levels in the leaf (Fig. 6). We propose that the 10 5B-Machi3-1 gene is similarly regulated in apple and transgenic tobacco plants (Fig. 7C). 11 During late phase of flower development, JA increases the MYB305 level in the nectary 12 and probably in the stigma, and then MYB305 binds to the 5 box region and promotes the 13 transcription of 5B-Machi3-1 gene. As Machi3-1 protein contains a signal peptide, it can 14 be secreted into the nectar explaining why 5B-Machi3-1 mRNA is abundant in the nectary, 15 while the Machi3-1 protein accumulates in the nectar. We hypothesize that Machi3-1 16 protein is also secreted from the stigma cells. As both apple and tobacco have wet stigma 17 (Sang et al., 2012;Losada and Herrero, 2012), we assume that in both plants Machi3-1 18 protein is secreted into the stigma exudate (Fig. 7C) (also see below). 19 Interestingly, the strong expression of MYB305 in nectary and stigma is not restricted to the 20 plants having wet stigma such as tobacco or apple. The Arabidopsis homolog MYB21 is 21 also expressed to high levels in both nectary and in the papilla cells of the dry stigma 22 (Osaka et al., 2013). 23 24

Machi3-1 protein might interfere with Erwinia infection at two different steps 25 26
We found that the Machi3-1 acidic chitinase III protein is present at very high 27 concentration in the nectar and the stigma of the 5B-Machi3-1 allele containing cultivars 28 ( Fig. 3 and 7). During apple infection, Erwinia propagates first in the stigma exudate, then 29 in the nectar and finally enters into the plant through the stomata of the nectary (Bubán et 30 al., 2003;Farkas et al., 2012). As biofilm formation is critical for the fruit and shoot biofilm formation of Erwinia at a concentration it is present in the nectar of 5B-Machi3-1 1 allele containing apple cultivars (Fig. 2). These findings suggest that the Machi3-1 protein 2 can interfere with the propagation and infection of Erwinia in the nectar of the 5B-Machi3-3 1 allele containing cultivars. Moreover, the Machi3-1 protein might also inhibit Erwinia 4 infection at the stigma. Machi3-1 was one of the most abundant protein in the 'Freedom' 5 stigma sample (Fig. 5D, see the + control sample and Fig. S3) that contains both the stigma 6 tissue and exudate. Machi3-1 has a signal peptide, therefore we assume that it is secreted 7 from the stigma cells and accumulates in the exudate. The Machi3-1 protein might be so 8 abundant in the stigma exudate that it can interfere with the propagation of Erwinia. Thus 9 we propose that accumulation of Machi3-1 protein could protect apples from Erwinia 10 infection by forming two barriers, it interferes with the replication and biofilm formation of 11 the Erwinia in the stigma exudate as well as in the nectar, thereby protecting the plants. 12 Our data shows that the 5B-Machi3-1 allele was introduced into different cultivars from M.         The protein profile of stigma samples of the same transgenic lines. 'Freedom' apple stigma extract was run as positive control (+cont.). * marks Machi3-1 band in the stigma samples.
Note that Machi3-1 protein is one of the most abundant protein in 'Freedom' stigma sample. Actin probe was used as loading control for the western-blot. Note that although stigma sample of 5B-Machi3-1 plant 6 is underloaded, the Machi3-1 protein is still easily detectable.