Synergistic approach of PCR-based fragment length analysis and amplicon deep sequencing reveals rich diversity of S-alleles in sweet cherries from the Caucasian region of origin.

Introduction: The self-incompatibility system in sweet cherry (Prunus avium L.) prevents fertilization with own or genetically related pollen, and is genetically determined by the multi-allelic S-locus. Therefore, determining S-alleles is crucial for plant breeding and fruit production, as it enables the selection of compatible combinations of S-genotypes for successful pollination. Methods: In this study, S-alleles were identified in a total of 260 genotypes from the Caucasian region, the species' center of origin. S-allele genotyping was conducted using PCR fragment length analysis with the standard marker PaConsI-F/R2 and reference genotypes, complemented by sequence analysis through amplicon deep sequencing. Results and discussion: The genotypes collected from Azerbaijan and Turkey exhibit a high allelic richness at the S-locus, particularly compared to modern sweet cherry cultivars worldwide. Nine previously undescribed S-alleles were identified and designated as S45, S46, S47, S48, S49, S50, S51, S52 and S53. Given the expected high diversity for other traits, this plant material represents a valuable resource for further breeding research and introgression of new traits in future breeding programs. Furthermore, our results underscore that fragment length alone may not be sufficient for unambiguous assignment of S-alleles due to minimal length differences between different alleles. To address this issue, an S-allele reference ladder was developed using the rich diversity for precise assignment of the S-alleles. This tool can be applied in future experiments as a robust and cost-effective method for accurate S-genotyping across different runs and laboratories. Additionally, several selected S-genotypes were planted in a trial field and will be maintained as an S-allele reference collection.

Recent Developments and Strategies for the Application of Agrobacterium-Mediated Transformation of Apple Malus × domestica Borkh.

Genetic transformation has become an important tool in plant genome research over the last three decades. This applies not only to model plants such as Arabidopsis thaliana but also increasingly to cultivated plants, where the establishment of transformation methods could still pose many problems. One of such plants is the apple (Malus spp.), the most important fruit of the temperate climate zone. Although the genetic transformation of apple using Agrobacterium tumefaciens has been possible since 1989, only a few research groups worldwide have successfully applied this technology, and efficiency remains poor. Nevertheless, there have been some developments, especially in recent years, which allowed for the expansion of the toolbox of breeders and breeding researchers. This review article attempts to summarize recent developments in the Agrobacterium-mediated transformation strategies of apple. In addition to the use of different tissues and media for transformation, agroinfiltration, as well as pre-transformation with a Baby boom transcription factor are notable successes that have improved transformation efficiency in apple. Further, we highlight targeted gene silencing applications. Besides the classical strategies of RNAi-based silencing by stable transformation with hairpin gene constructs, optimized protocols for virus-induced gene silencing (VIGS) and artificial micro RNAs (amiRNAs) have emerged as powerful technologies for silencing genes of interest. Success has also been achieved in establishing methods for targeted genome editing (GE). For example, it was recently possible for the first time to generate a homohistont GE line into which a biallelic mutation was specifically inserted in a target gene. In addition to these methods, which are primarily aimed at increasing transformation efficiency, improving the precision of genetic modification and reducing the time required, methods are also discussed in which genetically modified plants are used for breeding purposes. In particular, the current state of the rapid crop cycle breeding system and its applications will be presented.

Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis.

The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.

Evaluation of Scab and Mildew Resistance in the Gene Bank Collection of Apples in Dresden-Pillnitz.

A set of 680 apple cultivars from the Fruit Gene bank in Dresden Pillnitz was evaluated for the incidence of powdery mildew and scab in two consecutive years. The incidence of both scab and powdery mildew increased significantly in the second year. Sixty and 43 cultivars with very low incidence in both years of scab and powdery mildew, respectively, were analysed with molecular markers linked to known resistance genes. Thirty-five cultivars were identified to express alleles or combinations of alleles linked to Rvi2, Rvi4, Rvi6, Rvi13, Rvi14, or Rvi17. Twenty of them, modern as well as a few traditional cultivars known before the introduction or Rvi6 from Malus floribunda 821, amplified the 159 bp fragment of marker CH_Vf1 that is linked to Rvi6. Alleles linked to Pl1, Pld, or Plm were expressed from five cultivars resistant to powdery mildew. Eleven cultivars were identified to have very low susceptibility to both powdery mildew and scab. The information on resistance/susceptibility of fruit genetic resources towards economically important diseases is important for breeding and for replanting traditional cultivars. Furthermore, our work provides a well-defined basis for the discovery of undescribed, new scab, and powdery mildew resistance.

Transcriptional profile of AvrRpt2EA-mediated resistance and susceptibility response to Erwinia amylovora in apple.

Most of the commercial apple cultivars are highly susceptible to fire blight, which is the most devastating bacterial disease affecting pome fruits. Resistance to fire blight is described especially in wild Malus accessions such as M. × robusta 5 (Mr5), but the molecular basis of host resistance response to the pathogen Erwinia amylovora is still largely unknown. The bacterial effector protein AvrRpt2EA was found to be the key determinant of resistance response in Mr5. A wild type E. amylovora strain and the corresponding avrRpt2EA deletion mutant were used for inoculation of Mr5 to induce resistance or susceptible response, respectively. By comparison of the transcriptome of both responses, 211 differentially expressed genes (DEGs) were identified. We found that heat-shock response including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs) are activated in apple specifically in the susceptible response, independent of AvrRpt2EA. Further analysis on the expression progress of 81 DEGs by high-throughput real-time qPCR resulted in the identification of genes that were activated after inoculation with E. amylovora. Hence, a potential role of these genes in the resistance to the pathogen is postulated, including genes coding for enzymes involved in formation of flavonoids and terpenoids, ribosome-inactivating enzymes (RIPs) and a squamosa promoter binding-like (SPL) transcription factor.

The topoisomerase 3α zinc-finger domain T1 of Arabidopsis thaliana is required for targeting the enzyme activity to Holliday junction-like DNA repair intermediates.

Topoisomerase 3α, a class I topoisomerase, consists of a TOPRIM domain, an active centre and a variable number of zinc-finger domains (ZFDs) at the C-terminus, in multicellular organisms. Whereas the functions of the TOPRIM domain and the active centre are known, the specific role of the ZFDs is still obscure. In contrast to mammals where a knockout of TOP3α leads to lethality, we found that CRISPR/Cas induced mutants in Arabidopsis are viable but show growth retardation and meiotic defects, which can be reversed by the expression of the complete protein. However, complementation with AtTOP3α missing either the TOPRIM-domain or carrying a mutation of the catalytic tyrosine of the active centre leads to embryo lethality. Surprisingly, this phenotype can be overcome by the simultaneous removal of the ZFDs from the protein. In combination with a mutation of the nuclease AtMUS81, the TOP3α knockout proved to be also embryo lethal. Here, expression of TOP3α without ZFDs, and in particular without the conserved ZFD T1, leads to only a partly complementation in root growth-in contrast to the complete protein, that restores root length to mus81-1 mutant level. Expressing the E. coli resolvase RusA in this background, which is able to process Holliday junction (HJ)-like recombination intermediates, we could rescue this root growth defect. Considering all these results, we conclude that the ZFD T1 is specifically required for targeting the topoisomerase activity to HJ like recombination intermediates to enable their processing. In the case of an inactivated enzyme, this leads to cell death due to the masking of these intermediates, hindering their resolution by MUS81.

A Single Effector Protein, AvrRpt2EA, from Erwinia amylovora Can Cause Fire Blight Disease Symptoms and Induces a Salicylic Acid-Dependent Defense Response.

The AvrRpt2EA effector protein of Erwinia amylovora is important for pathogen recognition in the fire blight-resistant crabapple Malus × robusta 5; however, little is known about its role in susceptible apples. To study its function in planta, we expressed a plant-optimized version of AvrRpt2EA driven by a heat shock-inducible promoter in transgenic plants of the fire blight-susceptible cultivar Pinova. After induced expression of AvrRpt2EA, transgenic lines showed shoot necrosis and browning of older leaves, with symptoms similar to natural fire blight infections. Transgenic expression of this effector protein resulted in an increase in the expression of the salicylic acid (SA)-responsive PR-1 gene but, also, in the levels of SA and its derivatives, with diverse kinetics in leaves of different ages. In contrast, no increase of expression levels of VSP2 paralogs, used as marker genes for the activation of the jasmonic acid (JA)-dependent defense pathway, could be detected, which is in agreement with metabolic profiling of JA and its derivatives. Our work demonstrates that AvrRpt2EA acts as a virulence factor and induces the formation of SA and SA-dependent systemic acquired resistance.

The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana.

The stability of repetitive sequences in complex eukaryotic genomes is safeguarded by factors suppressing homologues recombination. Prominent in this is the role of the RTR complex. In plants, it consists of the RecQ helicase RECQ4A, the topoisomerase TOP3α and RMI1. Like mammals, but not yeast, plants harbor an additional complex partner, RMI2. Here, we demonstrate that, in Arabidopsis thaliana, RMI2 is involved in the repair of aberrant replication intermediates in root meristems as well as in intrastrand crosslink repair. In both instances, RMI2 is involved independently of the DNA helicase RTEL1. Surprisingly, simultaneous loss of RMI2 and RTEL1 leads to loss of male fertility. As both the RTR complex and RTEL1 are involved in suppression of homologous recombination (HR), we tested the efficiency of HR in the double mutant rmi2-2 rtel1-1 and found a synergistic enhancement (80-fold). Searching for natural target sequences we found that RTEL1 is required for stabilizing 45S rDNA repeats. In the double mutant with rmi2-2 the number of 45S rDNA repeats is further decreased sustaining independent roles of both factors in this process. Thus, loss of suppression of HR does not only lead to a destabilization of rDNA repeats but might be especially deleterious for tissues undergoing multiple cell divisions such as the male germline.

The RTR complex as caretaker of genome stability and its unique meiotic function in plants.

The RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RMI1 is involved in the processing of DNA recombination intermediates in all eukaryotes. In Arabidopsis thaliana the complex partners RECQ4A, topoisomerase 3α and RMI1 have been shown to be involved in DNA repair and in the suppression of homologous recombination in somatic cells. Interestingly, mutants of AtTOP3A and AtRMI1 are also sterile due to extensive chromosome breakage in meiosis I, a phenotype that seems to be specific for plants. Although both proteins are essential for meiotic recombination it is still elusive on what kind of intermediates they are acting on. Recent data indicate that the pattern of non-crossover (NCO)-associated meiotic gene conversion (GC) differs between plants and other eukaryotes, as less NCOs in comparison to crossovers (CO) could be detected in Arabidopsis. This indicates that NCOs happen either more rarely in plants or that the conversion tract length is significantly shorter than in other organisms. As the TOP3α/RMI1-mediated dissolution of recombination intermediates results exclusively in NCOs, we suggest that the peculiar GC pattern found in plants is connected to the unique role, members of the RTR complex play in plant meiosis.

Defining the roles of the N-terminal region and the helicase activity of RECQ4A in DNA repair and homologous recombination in Arabidopsis.

RecQ helicases are critical for the maintenance of genomic stability. The Arabidopsis RecQ helicase RECQ4A is the functional counterpart of human BLM, which is mutated in the genetic disorder Bloom's syndrome. RECQ4A performs critical roles in regulation of homologous recombination (HR) and DNA repair. Loss of RECQ4A leads to elevated HR frequencies and hypersensitivity to genotoxic agents. Through complementation studies, we were now able to demonstrate that the N-terminal region and the helicase activity of RECQ4A are both essential for the cellular response to replicative stress induced by methyl methanesulfonate and cisplatin. In contrast, loss of helicase activity or deletion of the N-terminus only partially complemented the mutant hyper-recombination phenotype. Furthermore, the helicase-deficient protein lacking its N-terminus did not complement the hyper-recombination phenotype at all. Therefore, RECQ4A seems to possess at least two different and independent sub-functions involved in the suppression of HR. By in vitro analysis, we showed that the helicase core was able to regress an artificial replication fork. Swapping of the terminal regions of RECQ4A with the closely related but functionally distinct helicase RECQ4B indicated that in contrast to the C-terminus, the N-terminus of RECQ4A was required for its specific functions in DNA repair and recombination.

The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis.

The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.