Application of new breeding techniques in fruit trees.

Climate change and rapid adaption of invasive pathogens pose a constant pressure on the fruit industry to develop improved varieties. Aiming to accelerate the development of better-adapted cultivars, new breeding techniques have emerged as a promising alternative to meet the demand of a growing global population. Accelerated breeding, cisgenesis, and CRISPR/Cas genome editing hold significant potential for crop trait improvement and have proven to be useful in several plant species. This review focuses on the successful application of these technologies in fruit trees to confer pathogen resistance and tolerance to abiotic stress and improve quality traits. In addition, we review the optimization and diversification of CRISPR/Cas genome editing tools applied to fruit trees, such as multiplexing, CRISPR/Cas-mediated base editing and site-specific recombination systems. Advances in protoplast regeneration and delivery techniques, including the use of nanoparticles and viral-derived replicons, are described for the obtention of exogenous DNA-free fruit tree species. The regulatory landscape and broader social acceptability for cisgenesis and CRISPR/Cas genome editing are also discussed. Altogether, this review provides an overview of the versatility of applications for fruit crop improvement, as well as current challenges that deserve attention for further optimization and potential implementation of new breeding techniques.

CRISPR/Cas9 genome-editing applied to MdPGT1 in apple results in reduced foliar phloridzin without impacting plant growth.

Phloridzin is the most abundant polyphenolic compound in apple (Malus × domestica Borkh.), which results from the action of a key phloretin-specific UDP-2'-O-glucosyltransferase (MdPGT1). Here, we simultaneously assessed the effects of targeting MdPGT1 by conventional transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing. To this end, we conducted transcriptomic and metabolic analyses of MdPGT1 RNA interference knockdown and genome-edited lines. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology, whereas genome-edited lines exhibited normal growth despite reduced foliar phloridzin. RNA-sequencing analysis identified a common core of regulated genes, involved in phenylpropanoid and flavonoid pathways. However, we identified genes and processes differentially modulated in stunted and genome-edited lines, including key transcription factors and genes involved in phytohormone signalling. Therefore, we conducted a phytohormone profiling to obtain insight into their role in the phenotypes observed. We found that salicylic and jasmonic acid were increased in dwarf lines, whereas auxin and ABA showed no correlation with the growth phenotype. Furthermore, bioactive brassinosteroids were commonly up-regulated, whereas gibberellin GA4 was distinctively altered, showing a sharp decrease in RNA interference knockdown lines. Expression analysis by reverse transcriptase-quantitative polymerase chain reaction expression analysis further confirmed transcriptional regulation of key factors involved in brassinosteroid and gibberellin interaction. These findings suggest that a differential modulation of phytohormones may be involved in the contrasting effects on growth following phloridzin reduction. The present study also illustrates how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple.

Insights into the cell-wall dynamics in grapevine berries during ripening and in response to biotic and abiotic stresses.

The cell wall (CW) is the dynamic structure of a plant cell, acting as a barrier against biotic and abiotic stresses. In grape berries, the modifications of pulp and skin CW during softening ensure flexibility during cell expansion and determine the final berry texture. In addition, the CW of grape berry skin is of fundamental importance for winemaking, controlling secondary metabolite extractability. Grapevine varieties with contrasting CW characteristics generally respond differently to biotic and abiotic stresses. In the context of climate change, it is important to investigate the CW dynamics occurring upon different stresses, to define new adaptation strategies. This review summarizes the molecular mechanisms underlying CW modifications during grapevine berry fruit ripening, plant-pathogen interaction, or in response to environmental stresses, also considering the most recently published transcriptomic data. Furthermore, perspectives of new biotechnological approaches aiming at modifying the CW properties based on other crops' examples are also presented.

The Role of Italy in the Use of Advanced Plant Genomic Techniques on Fruit Trees: State of the Art and Future Perspectives.

Climate change is deeply impacting the food chain production, lowering quality and yield. In this context, the international scientific community has dedicated many efforts to enhancing resilience and sustainability in agriculture. Italy is among the main European producers of several fruit trees; therefore, national research centers and universities undertook several initiatives to maintain the specificity of the 'Made in Italy' label. Despite their importance, fruit crops are suffering from difficulties associated with the conventional breeding approaches, especially in terms of financial commitment, land resources availability, and long generation times. The 'new genomic techniques' (NGTs), renamed in Italy as 'technologies for assisted evolution' (TEAs), reduce the time required to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop, the key genes and traits on which the scientific community is working, as well as the technological improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus is placed on the legal aspects in the European and in Italian contexts.

MdMTA-mediated m6 A modification enhances drought tolerance by promoting mRNA stability and translation efficiency of genes involved in lignin deposition and oxidative stress.

Although the N6 -methyladenosine (m6 A) modification is the most prevalent RNA modification in eukaryotes, the global m6 A modification landscape and its molecular regulatory mechanism in response to drought stress remain unclear. Transcriptome-wide m6 A methylome profiling revealed that m6 A is mainly enriched in the coding sequence and 3' untranslated region in response to drought stress in apple, by recognizing the plant-specific sequence motif UGUAH (H=A, U or C). We identified a catalytically active component of the m6 A methyltransferase complex, MdMTA. An in vitro methyl transfer assay, dot blot, LC-MS/MS and m6 A-sequencing (m6 A-seq) suggested that MdMTA is an m6 A writer and essential for m6 A mRNA modification. Further studies revealed that MdMTA is required for apple drought tolerance. m6 A-seq and RNA-seq analyses under drought conditions showed that MdMTA mediates m6 A modification and transcripts of mRNAs involved in oxidative stress and lignin deposition. Moreover, m6 A modification promotes mRNA stability and the translation efficiency of these genes in response to drought stress. Consistently, MdMTA enhances lignin deposition and scavenging of reactive oxygen species under drought conditions. Our results reveal the global involvement of m6 A modification in the drought response of perennial apple trees and illustrate its molecular mechanisms, thereby providing candidate genes for the breeding of stress-tolerant apple cultivars.

The regulatory module MdBT2-MdMYB88/MdMYB124-MdNRTs regulates nitrogen usage in apple.

Less than 40% of the nitrogen (N) fertilizer applied to soil is absorbed by crops. Thus, improving the N use efficiency of crops is critical for agricultural development. However, the underlying regulation of these processes remains largely unknown, particularly in woody plants. By conducting yeast two-hybrid assays, we identified one interacting protein of MdMYB88 and MdMYB124 in apple (Malus × domestica), namely BTB and TAZ domain protein 2 (MdBT2). Ubiquitination and protein stabilization analysis revealed that MdBT2 ubiquitinates and degrades MdMYB88 and MdMYB124 via the 26S proteasome pathway. MdBT2 negatively regulates nitrogen usage as revealed by the reduced fresh weight, dry weight, N concentration, and N usage index of MdBT2 overexpression calli under low-N conditions. In contrast, MdMYB88 and MdMYB124 increase nitrate absorption, allocation, and remobilization by regulating expression of MdNRT2.4, MdNRT1.8, MdNRT1.7, and MdNRT1.5 under N limitation, thereby regulating N usage. The results obtained illustrate the mechanism of a regulatory module comprising MdBT2-MdMYB88/MdMYB124-MdNRTs, through which plants modulate N usage. These data contribute to a molecular approach to improve the N usage of fruit crops under limited N acquisition.

Reduced fire blight susceptibility in apple cultivars using a high-efficiency CRISPR/Cas9-FLP/FRT-based gene editing system.

The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein MdDIPM4. In this work, MdDIPM4 knockout has been produced in two Malus × domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty-seven transgenic lines were screened to identify CRISPR/Cas9-induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss-of-function mutation were inoculated with the pathogen. Highly significant reduction in susceptibility was observed compared to control plants. Sequencing of five potential off-target sites revealed no mutation event. Moreover, our construct contained a heat-shock inducible FLP/FRT recombination system designed specifically to remove the T-DNA harbouring the expression cassettes for CRISPR/Cas9, the marker gene and the FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat-treated and screened by real-time PCR to quantify the exogenous DNA elimination. The T-DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9-FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DNA.

HIPM Is a Susceptibility Gene of Malus spp.: Reduced Expression Reduces Susceptibility to Erwinia amylovora.

Fire blight, a devastating disease caused by the bacterium Erwinia amylovora, is a major threat to apple crop production. To improve our understanding of the fire blight disease and to identify potential strategies to control the pathogen, we studied the apple protein HIPM (for HrpN-interacting protein from Malus spp.), which has previously been identified as interacting with the E. amylovora effector protein HrpN. Transgenic apple plants were generated with reduced HIPM expression, using an RNA interference construct, and were subsequently analyzed for susceptibility to E. amylovora infection. Lines exhibiting a greater than 50% silencing of HIPM expression showed a significant decrease in susceptibility to E. amylovora infection. Indeed, a correlation between HIPM expression and E. amylovora infection was identified, demonstrating the crucial role of HIPM during fire blight disease progression. Furthermore, an apple oxygen-evolving enhancer-like protein (MdOEE) was identified via a yeast two-hybrid screen to interact with HIPM. This result was confirmed with bimolecular fluorescence complementation assays and leads to new hypotheses concerning the response mechanism of the plant to E. amylovora as well as the mechanism of infection of the bacterium. These results suggest that MdOEE and, particularly, HIPM are promising targets for further investigations toward the genetic improvement of apple.

Adjusting the scent ratio: using genetically modified Vitis vinifera plants to manipulate European grapevine moth behaviour.

Herbivorous insects use olfactory cues to locate their host plant within a complex olfactory landscape. One such example is the European grapevine moth Lobesia botrana, a key pest of the grape in the Palearctic region, which recently expanded both its geographical and host plant range. Previous studies have showed that a synthetic blend of the three terpenoids, (E)-ß-caryophyllene, (E)-ß-farnesene and (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), was as attractive for the moth as the complete grape odour profile in laboratory conditions. The same studies also showed that the specific ratio of these compounds in the grape bouquet was crucial because a percentage variation in any of the three volatiles resulted in almost complete inhibition of the blend's attractiveness. Here, we report on the creation of stable grapevine transgenic lines, with modified (E)-ß-caryophyllene and (E)-ß-farnesene emission and thus with an altered ratio compared to the original plants. When headspace collections from these plants were tested in wind tunnel behavioural assays, they were less attractive than control extracts. This result was confirmed by testing synthetic blends imitating the ratio found on natural and transformed plants, as well as by testing the plants themselves. With this evidence, we suggest that a strategy based on volatile ratio modification may also interfere with the host-finding behaviour of L. botrana in the field, creating avenues for new pest control methods.

The knock-down of the expression of MdMLO19 reduces susceptibility to powdery mildew (Podosphaera leucotricha) in apple (Malus domestica).

Varieties resistant to powdery mildew (PM; caused by Podosphaera leucotricha) are a major component of sustainable apple production. Resistance can be achieved by knocking-out susceptibility S-genes to be singled out among members of the MLO (Mildew Locus O) gene family. Candidates are MLO S-genes of phylogenetic clade V up-regulated upon PM inoculation, such as MdMLO11 and 19 (clade V) and MdMLO18 (clade VII). We report the knock-down through RNA interference of MdMLO11 and 19, as well as the complementation of resistance with MdMLO18 in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 reduced PM disease severity by 75%, whereas the knock-down of MdMLO11, alone or in combination with MdMLO19, did not result in any reduction or additional reduction of susceptibility compared with MdMLO19 alone. The test in A. thaliana excluded a role for MdMLO18 in PM susceptibility. Cell wall appositions (papillae) were present in both PM-resistant and PM-susceptible plants, but were larger in resistant lines. No obvious negative phenotype was observed in plants with mlo genes knocked down. Apparently, MdMLO19 plays the pivotal role in apple PM susceptibility and its knock-down induces a very significant level of resistance.

Species-specific duplications driving the recent expansion of NBS-LRR genes in five Rosaceae species.

BACKGROUND: Disease resistance (R) genes from different Rosaceae species have been identified by map-based cloning for resistance breeding. However, there are few reports describing the pattern of R-gene evolution in Rosaceae species because several Rosaceae genome sequences have only recently become available. RESULTS: Since most disease resistance genes encode NBS-LRR proteins, we performed a systematic genome-wide survey of NBS-LRR genes between five Rosaceae species, namely Fragaria vesca (strawberry), Malus × domestica (apple), Pyrus bretschneideri (pear), Prunus persica (peach) and Prunus mume (mei) which contained 144, 748, 469, 354 and 352 NBS-LRR genes, respectively. A high proportion of multi-genes and similar Ks peaks (Ks = 0.1- 0.2) of gene families in the four woody genomes were detected. A total of 385 species-specific duplicate clades were observed in the phylogenetic tree constructed using all 2067 NBS-LRR genes. High percentages of NBS-LRR genes derived from species-specific duplication were found among the five genomes (61.81% in strawberry, 66.04% in apple, 48.61% in pear, 37.01% in peach and 40.05% in mei). Furthermore, the Ks and Ka/Ks values of TIR-NBS-LRR genes (TNLs) were significantly greater than those of non-TIR-NBS-LRR genes (non-TNLs), and most of the NBS-LRRs had Ka/Ks ratios less than 1, suggesting that they were evolving under a subfunctionalization model driven by purifying selection. CONCLUSIONS: Our results indicate that recent duplications played an important role in the evolution of NBS-LRR genes in the four woody perennial Rosaceae species. Based on the phylogenetic tree produced, it could be inferred that species-specific duplication has mainly contributed to the expansion of NBS-LRR genes in the five Rosaceae species. In addition, the Ks and Ka/Ks ratios suggest that the rapidly evolved TNLs have different evolutionary patterns to adapt to different pathogens compared with non-TNL resistant genes.

Genetically engineered Thompson Seedless grapevine plants designed for fungal tolerance: selection and characterization of the best performing individuals in a field trial.

The fungi Botrytis cinerea and Erysiphe necator are responsible for gray mold and powdery mildew diseases, respectively, which are among the most devastating diseases of grapes. Two endochitinase (ech42 and ech33) genes and one N-acetyl-ß-D-hexosaminidase (nag70) gene from biocontrol agents related to Trichoderma spp. were used to develop a set of 103 genetically modified (GM) 'Thompson Seedless' lines (568 plants) that were established in open field in 2004 and evaluated for fungal tolerance starting in 2006. Statistical analyses were carried out considering transgene, explant origin, and plant response to both fungi in the field and in detached leaf assays. The results allowed for the selection of the 19 consistently most tolerant lines through two consecutive years (2007-2008 and 2008-2009 seasons). Plants from these lines were grafted onto the rootstock Harmony and established in the field in 2009 for further characterization. Transgene status was shown in most of these lines by Southern blot, real-time PCR, ELISA, and immunostrips; the most tolerant candidates expressed the ech42-nag70 double gene construct and the ech33 gene from a local Hypocrea virens isolate. B. cinerea growth assays in Petri dishes supplemented with berry juices extracted from the most tolerant individuals of the selected population was inhibited. These results demonstrate that improved fungal tolerance can be attributed to transgene expression and support the iterative molecular and physiological phenotyping in order to define selected individuals from a population of GM grapevines.

Characterization of the MLO gene family in Rosaceae and gene expression analysis in Malus domestica.

BACKGROUND: Powdery mildew (PM) is a major fungal disease of thousands of plant species, including many cultivated Rosaceae. PM pathogenesis is associated with up-regulation of MLO genes during early stages of infection, causing down-regulation of plant defense pathways. Specific members of the MLO gene family act as PM-susceptibility genes, as their loss-of-function mutations grant durable and broad-spectrum resistance. RESULTS: We carried out a genome-wide characterization of the MLO gene family in apple, peach and strawberry, and we isolated apricot MLO homologs through a PCR-approach. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Homologs that are candidates for being PM susceptibility genes were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen Podosphaera leucotricha. CONCLUSIONS: Genomic tools available for Rosaceae were exploited in order to characterize the MLO gene family. Candidate MLO susceptibility genes were identified. In follow-up studies it can be investigated whether silencing or a loss-of-function mutations in one or more of these candidate genes leads to PM resistance.

Calcium imaging perspectives in plants.

The calcium ion (Ca2+) is a versatile intracellular messenger. It provides dynamic regulation of a vast array of gene transcriptions, protein kinases, transcription factors and other complex downstream signaling cascades. For the past six decades, intracellular Ca2+ concentration has been significantly studied and still many studies are under way. Our understanding of Ca2+ signaling and the corresponding physiological phenomenon is growing exponentially. Here we focus on the improvements made in the development of probes used for Ca2+ imaging and expanding the application of Ca2+ imaging in plant science research.

Assessment and Partial Characterization of Candidate Genes in Dihydrochalcone and Arbutin Biosynthesis in an Apple-Pear Hybrid by De Novo Transcriptome Assembly.

Apples (Malus × domestica Borkh.) and pears (Pyrus communis L.) are valuable crops closely related within the Rosaceae family with reported nutraceutical properties derived from secondary metabolites including phloridzin and arbutin, which are distinctive phenolic metabolites characterizing apples and pears, respectively. Here, we generated a de novo transcriptome assembly of an intergeneric hybrid between apple and pear, accumulating intermediate levels of phloridzin and arbutin. Combining RNA-seq, in silico functional annotation prediction, targeted gene expression analysis, and expression-metabolite correlations, we identified candidate genes for functional characterization, resulting in the identification of active arbutin synthases in the hybrid and parental genotypes. Despite exhibiting an active arbutin synthase in vitro, the natural lack of arbutin in apples is reasoned by the absence of the substrate and broad substrate specificity. Altogether, our study serves as the basis for future assessment of potential physiological roles of identified genes by genome editing of hybrids and pears.

The de novo, chromosome-level genome assembly of the sweet chestnut (Castanea sativa Mill.) Cv. Marrone Di Chiusa Pesio.

OBJECTIVES: The sweet chestnut Castanea sativa Mill. is the only native Castanea species in Europe, and it is a tree of high economic value that provides appreciated fruits and valuable wood. In this study, we assembled a high-quality nuclear genome of the ancient Italian chestnut variety 'Marrone di Chiusa Pesio' using a combination of Oxford Nanopore Technologies long reads, whole-genome and Omni-C Illumina short reads. DATA DESCRIPTION: The genome was assembled into 238 scaffolds with an N50 size of 21.8 Mb and an N80 size of 7.1 Mb for a total assembled sequence of 750 Mb. The BUSCO assessment revealed that 98.6% of the genome matched the embryophyte dataset, highlighting good completeness of the genetic space. After chromosome-level scaffolding, 12 chromosomes with a total length of 715.8 and 713.0 Mb were constructed for haplotype 1 and haplotype 2, respectively. The repetitive elements represented 37.3% and 37.4% of the total assembled genome in haplotype 1 and haplotype 2, respectively. A total of 57,653 and 58,146 genes were predicted in the two haplotypes, and approximately 73% of the genes were functionally annotated using the EggNOG-mapper. The assembled genome will be a valuable resource and reference for future chestnut breeding and genetic improvement.

A genome-wide phylogenetic reconstruction of family 1 UDP-glycosyltransferases revealed the expansion of the family during the adaptation of plants to life on land.

For almost a decade, our knowledge on the organisation of the family 1 UDP-glycosyltransferases (UGTs) has been limited to the model plant A. thaliana. The availability of other plant genomes represents an opportunity to obtain a broader view of the family in terms of evolution and organisation. Family 1 UGTs are known to glycosylate several classes of plant secondary metabolites. A phylogeny reconstruction study was performed to get an insight into the evolution of this multigene family during the adaptation of plants to life on land. The organisation of the UGTs in the different organisms was also investigated. More than 1500 putative UGTs were identified in 12 fully sequenced and assembled plant genomes based on the highly conserved PSPG motif. Analyses by maximum likelihood (ML) method were performed to reconstruct the phylogenetic relationships existing between the sequences. The results of this study clearly show that the UGT family expanded during the transition from algae to vascular plants and that in higher plants the clustering of UGTs into phylogenetic groups appears to be conserved, although gene loss and gene gain events seem to have occurred in certain lineages. Interestingly, two new phylogenetic groups, named O and P, that are not present in A. thaliana were discovered.

Differential expression of CPKs and cytosolic Ca2+ variation in resistant and susceptible apple cultivars (Malus x domestica) in response to the pathogen Erwinia amylovora and mechanical wounding.

BACKGROUND: Plant calcium (Ca2+) signals are involved in a wide array of intracellular signalling pathways following pathogen invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate signalling following Ca2+ influx after pathogen infection. However, to date this prediction has remained elusive. RESULTS: We conducted a genome-wide identification of the Malus x domestica CPK (MdCPK) gene family and identified 30 CPK genes. Comparative phylogenetic analysis of Malus CPKs with CPKs of Arabidopsis thaliana (AtCPKs), Oryza sativa (OsCPKs), Populous trichocarpa (PtCPKs) and Zea mays (ZmCPKs) revealed four different groups. From the phylogenetic tree, we found that MdCPKs are closely related to AtCPKs and PtCPKs rather than OsCPKs and ZmCPKs, indicating their dicot-specific origin. Furthermore, comparative quantitative real time PCR and intracellular cytosolic calcium ([Ca2+]cyt) analysis were carried out on fire blight resistant and susceptible M. x domestica apple cultivars following infection with a pathogen (Erwinia amylovora) and/or mechanical damage. Calcium analysis showed an increased [Ca2+]cyt over time in resistant cultivars as compared to susceptible cultivars. Gene expression studies showed that 11 out of the 30 MdCPKs were differentially expressed following pathogen infection. CONCLUSIONS: We studied the genome-wide analysis of MdCPK gene family in Malus x domestica and analyzed their differential gene expression along with cytosolic calcium variation upon pathogen infection. There was a striking difference in MdCPKs gene expressions and [Ca2+]cyt variations between resistant and susceptible M. x domestica cultivars in response to E. amylovora and mechanical wounding. Our genomic and bioinformatic analysis provided an important insight about the role of MdCPKs in modulating defence responses in susceptible and resistant apple cultivars. It also provided further information on early signalling and downstream signalling cascades in response to pathogenic and mechanical stress.