Transcriptome analysis of transgenic apple fruit overexpressing microRNA172 reveals candidate transcription factors regulating apple fruit development at early stages

Background MicroRNA172 (miR172) has been proven to be critical for fruit growth, since elevated miR172 activity blocks the growth of apple (Malus x domestica Borkh.) fruit. However, it is not clear how overexpression of miR172 affects apple fruit developmental processes. Methods To answer this question, the present study, analyzed global transcriptional changes in miR172-overexpressing (miR172OX) and nongenetically modified wild-type (WT) apple fruit at two developmental stages and in different fruit tissues via RNA-seq. In addition, two cultivars, ‘Hanfu’ and ‘M9’, which have naturally fruit size variation, were included to identify miR172-dependent DEGs. qRT–PCRwas used to verify the reliability of our RNA-seq data. Results Overexpression of miR172 altered the expression levels of many cell proliferation- and cell expansion-related genes. Twenty-four libraries were generated, and 10,338 differentially expressed genes (DEGs) were detected between miR172OX and WT fruit tissues. ‘Hanfu’ and ‘M9’ are two common cultivars that bear fruit of different sizes (250 g and 75 g, respectively). Six libraries were generated, and 3,627 DEGs were detected between ‘Hanfu’ and ‘M9’. After merging the two datasets, 6,888 candidate miR172-specific DEGs were identified. The potential networks associated with fruit size triggered traits were defined among genes belonging to the families of hormone synthesis, signaling pathways, and transcription factors. Our comparative transcriptome analysis provides insights into transcriptome responses to miR172 overexpression in apple fruit and a valuable database for future studies to validate functional genes and elucidate the fruit developmental mechanisms in apple.

Successful intergeneric transfer of a major apple scab resistance gene (Rvi6) from apple to pear and precise comparison of the downstream molecular mechanisms of this resistance in both species

Background Scab is the most important fungal disease of apple and pear. Apple (Malus x domestica Borkh.) and European pear (Pyrus communis L.) are genetically related but they are hosts of two different fungal species: Venturia inaequalis for apple and V. pyrina for European pear. The apple/V. inaequalis pathosystem is quite well known, whereas knowledge about the pear/V. pyrina pathosystem is still limited. The aim of our study was to analyse the mode of action of a major resistance gene of apple (Rvi6) in transgenic apple and pear plants interacting with the two scab species (V. inaequalis and V. pyrina), in order to determine the degree of functional transferability between the two pathosystems. Results Transgenic pear clones constitutively expressing the Rvi6 gene from apple were compared to a scab transgenic apple clone carrying the same construct. After inoculation in greenhouse with V. pyrina, strong defense reactions and very limited sporulation were observed on all transgenic pear clones tested. Microscopic observations revealed frequent aborted conidiophores in the Rvi6 transgenic pear / V. pyrina interaction. The macro- and microscopic observations were very comparable to the Rvi6 apple / V. inaequalis interaction. However, this resistance in pear proved variable according to the strain of V. pyrina, and one of the strains tested overcame the resistance of most of the transgenic pear clones. Comparative transcriptomic analyses of apple and pear resistant interactions with V. inaequalis and V. pyrina, respectively, revealed different cascades of molecular mechanisms downstream of the pathogen recognition by Rvi6 in the two species. Signal transduction was triggered in both species with calcium (and G-proteins in pear) and interconnected hormonal signaling (jasmonic acid in pear, auxins in apple and brassinosteroids in both species), without involvement of salicylic acid. This led to the induction of defense responses such as a remodeling of primary and secondary cell wall, lipids biosynthesis (galactolipids in apple and cutin and cuticular waxes in pear), systemic acquired resistance signal generation (in apple) or perception in distal tissues (in pear), and the biosynthesis of phenylpropanoids (flavonoids in apple but also lignin in pear). Conclusion This study is the first example of a successful intergeneric transfer of a resistance gene among Rosaceae, with a resistance gene functioning towards another species of pathogen.

MdWRKY75e enhances resistance to Alternaria alternata in Malus domestica

The Alternaria alternata apple pathotype adversely affects apple (Malus domestica Borkh.) cultivation. However, the molecular mechanisms underlying enhanced resistance to this pathogen in apple remain poorly understood. We have previously reported that MdWRKY75 expression is upregulated by A. alternata infection in ‘Sushuai’ apples. In this study, we discovered that overexpression of MdWRKY75e increased the resistance of transgenic apple lines to A. alternata infection, whereas silencing this gene enhanced susceptibility to A. alternata infection. Furthermore, we found that MdWRKY75e directly binds to the MdLAC7 promoter to regulate the biosynthesis of laccase and increase the biosynthesis of lignin during A. alternata infection. Moreover, the thickening of the cell wall enhanced the mechanical defense capabilities of apple. In addition, we found that jasmonic acid remarkably induced MdWRKY75e expression, and its levels in transgenic apple lines were elevated. These results indicate that MdWRKY75e confers resistance to the A. alternata apple pathotype mainly via the jasmonic acid pathway and that pathogenesis-related genes and antioxidant-related enzyme activity are involved in the disease resistance of MdWRKY75e transgenic plants. In conclusion, our findings provide insights into the importance of MdWRKY75e for resistance to A. alternata infection in apples.

MdVQ37 overexpression reduces basal thermotolerance in transgenic apple by affecting transcription factor activity and salicylic acid homeostasis

High temperature (HT) is one of the most important environmental stress factors and seriously threatens plant growth, development, and production. VQ motif-containing proteins are transcriptional regulators that have been reported to regulate plant growth and developmental processes, including responses to biotic and abiotic stresses. However, the relationships between VQ motif-containing proteins and HT stress have not been studied in depth in plants. In this study, transgenic apple (Malus domestica) plants overexpressing the apple VQ motif-containing protein-coding gene (MdVQ37) were exposed to HT stress, and the transgenic lines exhibited a heat-sensitive phenotype. In addition, physiological and biochemical studies revealed that, compared with WT plants, transgenic lines had lower enzymatic activity and photosynthetic capacity and lower amounts of nonenzymatic antioxidant system metabolites under HT stress. Transcriptome analysis revealed 1379 genes whose expression differed between the transgenic lines and WT plants. GO and KEGG pathway analyses showed that transcription factor activity and plant hormone signaling pathways were differentially influenced and enriched in the transgenic lines. Salicylic acid (SA) content analysis indicated that overexpression of MdVQ37 reduced the content of endogenous SA by regulating the expression of SA catabolism-related genes, which ultimately resulted in disruption of the SA-dependent signaling pathway under HT stress. The application of SA slightly increased the survival rate of the transgenic lines under HT stress. Taken together, our results indicate that apple MdVQ37 has a regulatory function in basal thermotolerance by modulating the activity of transcription factors and SA homeostasis. Overall, this study provides novel insights that improve our understanding of the various functions of VQ motif-containing proteins.

No Evidence of Unexpected Transgenic Insertions in T1190 – A Transgenic Apple Used in Rapid Cycle Breeding – Following Whole Genome Sequencing

Rapid cycle breeding uses transgenic early flowering plants as crossbreed parents to facilitate the shortening of breeding programs for perennial crops with long-lasting juvenility. Rapid cycle breeding in apple was established using the transgenic genotype T1190 expressing the BpMADS4 gene of silver birch. In this study, the genomes of T1190 and its non-transgenic wild-type PinS (F1-offspring of ‘Pinova’ and ‘Idared’) were sequenced by Illumina short-read sequencing in two separate experiments resulting in a mean sequencing depth of 182× for T1190 and 167× for PinS. The sequencing revealed 8,450 reads, which contain sequences of ≥20 bp identical to the plant transformation vector. These reads were assembled into 125 contigs, which were examined to see whether they contained transgenic insertions or if they are not using a five-step procedure. The sequence of one contig represents the known T-DNA insertion on chromosome 4 of T1190. The sequences of the remaining contigs were either equally present in T1190 and PinS, their part with sequence identity to the vector was equally present in apple reference genomes, or they seem to result from endophytic contaminations rather than from additional transgenic insertions. Therefore, we conclude that the transgenic apple plant T1190 contains only one transgenic insertion, located on chromosome 4, and shows no further partial insertions of the transformation vector.Accession Numbers: JQ974028.1.

Successful intergeneric transfer of a major apple scab resistance gene (Rvi6) from apple to pear and precise comparison of the downstream molecular mechanisms of this resistance in both species

Background: Scab is the most important fungal disease of apple and pear. Apple (Malus x domestica Borkh.) and European pear (Pyrus communis L.) are genetically related but they are hosts of two different fungal species: Venturia inaequalis for apple and V. pyrina for European pear. The apple/V. inaequalis pathosystem is quite well known, whereas knowledge about the pear/V. pyrina pathosystem is still limited. The aim of our study was to analyse the mode of action of a major resistance gene of apple (Rvi6) in transgenic apple and pear plants interacting with the two scab species (V. inaequalis and V. pyrina), in order to determine the degree of functional transferability between the two pathosystems. Results: Transgenic pear clones constitutively expressing the Rvi6 gene from apple were compared to a scab transgenic apple clone carrying the same construct. After inoculation in greenhouse with V. pyrina, strong defense reactions and very limited sporulation were observed on all transgenic pear clones tested. Microscopic observations revealed frequent aborted conidiophores in the Rvi6 transgenic pear / V. pyrina interaction. The macro- and microscopic observations were very comparable to the Rvi6 apple / V. inaequalis interaction. However, this resistance in pear proved variable according to the strain of V. pyrina, and one of the strains tested overcame the resistance of most of the transgenic pear clones. Comparative transcriptomic analyses of apple and pear resistant interactions with V. inaequalis and V. pyrina, respectively, revealed different cascades of molecular mechanisms downstream of the pathogen recognition by Rvi6 in the two species. Signal transduction was triggered in both species with calcium (and G-proteins in pear) and interconnected hormonal signaling (jasmonic acid in pear, auxins in apple and brassinosteroids in both species), without involvement of salicylic acid. This led to the induction of defense responses such as a remodeling of primary and secondary cell wall, lipids biosynthesis (galactolipids in apple and cutin and cuticular waxes in pear), systemic acquired resistance signal generation (in apple) or perception in distal tissues (in pear), and the biosynthesis of phenylpropanoids (flavonoids in apple but also lignin in pear). Conclusion: This study is the first example of a successful intergeneric transfer of a resistance gene among Rosaceae, with a resistance gene functioning towards another species of pathogen.

Exogenous application of xanthine and uric acid and nucleobase-ascorbate transporter MdNAT7 expression regulate salinity tolerance in apple

Background Soil salinity is a critical threat to global agriculture. In plants, the accumulation of xanthine activates xanthine dehydrogenase (XDH), which catalyses the oxidation/conversion of xanthine to uric acid to remove excess reactive oxygen species (ROS). The nucleobase-ascorbate transporter (NAT) family is also known as the nucleobase-cation symporter (NCS) or AzgA-like family. NAT is known to transport xanthine and uric acid in plants. The expression of MdNAT is influenced by salinity stress in apple. Results In this study, we discovered that exogenous application of xanthine and uric acid enhanced the resistance of apple plants to salinity stress. In addition, MdNAT7 overexpression transgenic apple plants showed enhanced xanthine and uric acid concentrations and improved tolerance to salinity stress compared with nontransgenic plants, while opposite phenotypes were observed for MdNAT7 RNAi plants. These differences were probably due to the enhancement or impairment of ROS scavenging and ion homeostasis abilities. Conclusion Our results demonstrate that xanthine and uric acid have potential uses in salt stress alleviation, and MdNAT7 can be utilized as a candidate gene to engineer resistance to salt stress in plants.