Identification and mapping of the novel apple scab resistance gene Vd3

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most devastating diseases for the apple growing in temperate zones with humid springs and summers. Breeding programs around the world have been able to identify several sources of resistance, the Vf from Malus floribunda 821 being the most frequently used. The appearance of two new races of V. inaequalis (races 6 and 7) in several European countries that are able to overcome the resistance of the Vf gene put in evidence the necessity of the combination of different resistance genes in the same genotype (pyramiding). Here, we report the identification and mapping of a new apple scab resistance gene (Vd3) from the resistant selection “1980-015-25” of the apple breeding program at Plant Research International, The Netherlands. This selection contains also the Vf gene and the novel V25 gene for apple scab resistance. We mapped Vd3 on linkage group 1, 1 cM to the south of Vf in repulsion phase to it. Based on pedigree analysis and resistance tests, it could be deduced that 1980-015-25 had inherited Vd3 from the founder “D3.” This gene provides resistance to the highly virulent EU-NL-24 strain of race 7 of V. inaequalis capable of overcoming the resistance from Vf and Vg. JMS and SGJ contributed equally to this work     

Functional analysis and expression profiling of <Emphasis Type="Italic">HcrVf1</Emphasis> and <Emphasis Type="Italic">HcrVf2</Emphasis> for development of scab resistant cisgenic and intragenic apples

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (P_MdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana’.     

Cas9HF1 enhanced specificity in Ustilago maydis

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely used as a tool to precisely manipulate genomic sequence targeted by sgRNA (single guide RNA) and is adapted in different species for genome editing. One of the major concerns of CRISPR-Cas9 is the possibility of off-target effects, which can be remedied by the deployment of high fidelity Cas9 variants. Ustilago maydis is a maize fungal pathogen, which has served as a model organism for biotrophic pathogens for decades. The successful adaption of CRISPR-Cas9 in U. maydis greatly facilitated effector biology studies. Here, we constructed an U. maydis reporter strain that allows in vivo quantification of efficiency and target specificity of three high fidelity Cas9 variants, Cas9HF1, Cas9esp1.1 and Cas9hypa. This approach identified Cas9HF1 as most specific Cas9 variant in U. maydis. Furthermore, whole genome sequencing showed absence of off-target effects in U. maydis by CRISPR-Cas9 editing.     

Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon

Research on CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated protein) systems has led to the revolutionary CRISPR/Cas9 genome editing technique. However, for most archaea and half of bacteria, exploitation of their native CRISPR-Cas machineries may be more straightforward and convenient. In this study, we harnessed the native type I-B CRISPR-Cas system for precise genome editing in the polyploid haloarchaeon Haloarcula hispanica. After testing different designs, the editing tool was optimized to be a single plasmid that carries both the self-targeting mini-CRISPR and a 600–800 bp donor. Significantly, chromosomal modifications, such as gene deletion, gene tagging or single nucleotide substitution, were precisely introduced into the vast majority of the transformants. Moreover, we showed that simultaneous editing of two genomic loci could also be readily achieved by one step. In summary, our data demonstrate that the haloarchaeal CRISPR-Cas system can be harnessed for genome editing in this polyploid archaeon, and highlight the convenience and efficiency of the native CRISPR-based genome editing strategy.     

Mapping and functional analysis of four apple receptor-like protein kinases related to <Emphasis Type="Italic">LRPKm1</Emphasis> in <Emphasis Type="Italic">HcrVf2</Emphasis>-transgenic and wild-type apple plants

The Malus–Venturia inaequalis interaction is the most studied plant–pathogen interaction involving a woody species. Besides the cloning of an apple scab resistance gene HcrVf2, several sequences have been recently identified that are modulated after pathogen recognition in Vf-resistant genotypes. Among these, there is a putative leucine-rich repeat receptor-like protein kinase from the apple scab-resistant cv. Florina, named LRPKm1 that is induced after V. inaequalis inoculation and salicylic acid treatment. In this work, the isolation, characterization, and mapping of four new genes belonging to the LRPKm multigene family are reported. According to their cumulative expression profiles in HcrVf2-transgenic and wild-type apple plants treated with V. inaequalis, LRPKm genes have been divided in two groups. LRPKm1 and LRPKm3, giving a response related to the presence of HcrVf2, are probably involved in the recognition of pathogen-derived signals. LRPKm2 and LRPKm4, with an expression profile unrelated to the HcrVf2 gene, are putatively involved in the plant basal defense. Furthermore, we have localized LRPKm proteins at the cytological level in the plasma membrane of epidermal cells in resistant genotypes following pathogen challenge, thus confirming software predictions and molecular results. The possible involvement of LRPKm proteins in apple scab resistance and in the plant basal defense makes them attractive for a better comprehension of the molecular mechanisms of the signal transduction pathways after pathogen recognition.     

CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production

Cyanobacteria hold promise as a cell factory for producing biofuels and bio-derived chemicals, but genome engineering of cyanobacteria such as Synechococcus elongatus PCC 7942 poses challenges because of their oligoploidy nature and long-term instability of the introduced gene. CRISPR-Cas9 is a newly developed RNA-guided genome editing system, yet its application for cyanobacteria engineering has yet to be reported. Here we demonstrated that CRISPR-Cas9 system can effectively trigger programmable double strand break (DSB) at the chromosome of PCC 7942 and provoke cell death. With the co-transformation of template plasmid harboring the gene cassette and flanking homology arms, CRISPR-Cas9-mediated DSB enabled precise gene integration, ameliorated the homologous recombination efficiency and allowed the use of lower amount of template DNA and shorter homology arms. The CRISPR-Cas9-induced cell death imposed selective pressure and enhanced the chance of concomitant integration of gene cassettes into all chromosomes of PCC 7942, hence accelerating the process of obtaining homogeneous and stable recombinant strains. We further explored the feasibility of engineering cyanobacteria by CRISPR-Cas9-assisted simultaneous glgc knock-out and gltA/ppc knock-in, which improved the succinate titer to 435.0±35.0 μg/L, an ≈11-fold increase when compared with that of the wild-type cells. These data altogether justify the use of CRISPR-Cas9 for genome engineering and manipulation of metabolic pathways in cyanobacteria.     

Phloridzin and apple scab

Phloridzin, sieboldin, trilobatin, phloretin and 3-hydroxyphloretin can all be used as carbon sources by Venturia inaequalis in culture. Resistance to apple scab was not linked with inheritance of sieboldin or trilobatin in seedlings. There is no direct connection between phloridzin or its breakdown products and scab resistance.     

Construction and characterization of a bacterial artificial chromosome library of apple

 A bacterial artificial chromosome (BAC) library has been constructed from apple (Malus×domestica Borkh.) using the variety “Florina”, which is resistant to scab (Venturia inaequalis) by virtue of the Vf gene. Since apple leaves are rich in polyphenols, high-molecular-weight DNA was extracted from leaf nuclei with a protocol adapted to apple. The nuclei were then embedded in agarose microbeads and partially digested by varying ratios of EcoRI to EcoRI methylase. The resulting DNA fragments were size-selected by pulsed-field gel electrophoresis, ligated to the BAC cloning vector pECBAC1 and transformed into Escherichia coli cells by electroporation. A total of 36 864 recombinant clones (BACs) were obtained. The library has an average insert size of 120 kb and represents approximately 5×apple haploid-genome equivalents. It was screened with six cDNA probes using the chemiluminescent DIG system. An average of 4.4 clones was detected for each locus. The apple BAC library will be used to isolate the Vf scab resistance gene through map-based cloning. In this connection the library was screened with a marker closely linked to the Vf gene and six positive clones have been isolated. This library should thus be well suited for map-based gene cloning, in particular for the isolation of the Vf gene and for the construction of a physical map of the apple genome. Received: 19 February 1998 / Accepted: 30 April 1998     

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc^−/−) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.     

Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase

The CRISPR-Cas9 system is a powerful and revolutionary genome-editing tool for eukaryotic genomes, but its use in bacterial genomes is very limited. Here, we investigated the use of the Streptococcus pyogenes CRISPR-Cas9 system in editing the genome of Clostridium cellulolyticum, a model microorganism for bioenergy research. Wild-type Cas9-induced double-strand breaks were lethal to C. cellulolyticum due to the minimal expression of nonhomologous end joining (NHEJ) components in this strain. To circumvent this lethality, Cas9 nickase was applied to develop a single-nick-triggered homologous recombination strategy, which allows precise one-step editing at intended genomic loci by transforming a single vector. This strategy has a high editing efficiency (>95%) even using short homologous arms (0.2 kb), is able to deliver foreign genes into the genome in a single step without a marker, enables precise editing even at two very similar target sites differing by two bases preceding the seed region, and has a very high target site density (median interval distance of 9 bp and 95.7% gene coverage in C. cellulolyticum). Together, these results establish a simple and robust methodology for genome editing in NHEJ-ineffective prokaryotes.     

CRISPR-Cas9 base editors and their current role in human therapeutics

BackgroundConsistent progress has been made to create more efficient and useful CRISPR-Cas9-based molecular toolsfor genomic modification.MethodsThis review focuses on recent articles that have employed base editors (BEs) for both clinical and research purposes.ResultsCRISPR-Cas9 BEs are a useful system because of their highefficiency and broad applicability to gene correction and disruption. In addition, base editing has beensuggested as a safer approach than other CRISPR-Cas9-based systems, as it limits double-strand breaksduring multiplex gene knockout and does not require a toxic DNA donor molecule for genetic correction.ConclusionAs such, numerous industry and academic groups are currently developing base editing strategies withclinical applications in cancer immunotherapy and gene therapy, which this review will discuss, with a focuson current and future applications of in vivo BE delivery.     

Expression profiling in HcrVf2-transformed apple plants in response to Venturia inaequalis

Apple scab resistance is one of the most well-characterized plant–pathogen interactions in a woody plant species. While the HcrVf2 gene from the wild apple Malus floribunda 821 has proved capable of conferring scab resistance to the susceptible cv. Gala after genetic transformation, its identification represents only the first step in understanding the molecular mechanisms and, hence, the network of genes underlying the defence response. We used a PCR-based suppression subtractive hybridization to identify apple genes that are differentially expressed after Venturia inaequalis inoculation. Subtractive hybridization was performed between cDNA from challenged leaves of HcrVf2-resistant transgenic Gala and susceptible cv. Gala plants. A library of 523 unigenes was constructed and characterized by assigning a putative function via comparison with public databases. This set of pathogen-modulated apple genes includes many defence-related genes and is therefore an important source of information for understanding the molecular basis of the Malus–V. inaequalis interaction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering.     

CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry

To develop an effective genome editing tool for blueberry breeding, CRISPR-Cas9 and CRISPR-Cas12a were evaluated for their editing efficiencies of a marker gene, beta-glucuronidase (gusA), which was previously introduced into two blueberry cultivars each a single-copy transgene. Four expression vectors were built, with CRISPR-Cas9 and CRISPR-Cas12a each driven by a 35S promoter or AtUbi promoter. Each vector contained two editing sites in the gusA. These four vectors were respectively transformed into the leaf explants of transgenic gusA blueberry and the resulting transgenic calli were induced under hygromycin selection. GUS staining showed that some small proportions of the hygromycin-resistant calli had non-GUS stained sectors, suggesting some possible occurrences of gusA editing. We sequenced GUS amplicons spanning the two editing sites in three blueberry tissues and found about 5.5% amplicons having editing features from the calli transformed with the 35S-Cas9 vector. Further, we conducted a second round of shoot regeneration from leaf explants derived from the initial Cas9- and Cas12a-containing calli (T₀) and analyzed amplicons of the target editing region. Of the newly induced shoots, 15.5% for the 35S-Cas9 and 5.3% for the AtUbi-Cas9 showed non-GUS staining, whereas all of the shoots containing the Cas12a vectors showed blue staining. Sanger sequencing confirmed the editing-induced mutations in two representative non-GUS staining lines. Clearly, the second round of regeneration had enriched editing events and enhanced the production of edited shoots. The results and protocol described will be helpful to facilitating high-precision breeding of blueberries using CRISPR Cas technologies.

     

The Vh2 and Vh4 scab resistance genes in two differential hosts derived from Russian apple R12740-7A map to the same linkage group of apple

Russian apple R12740-7A is the designation for an accession grown from seed collected in Russia, which was found to be highly resistant to apple scab. The resistance has historically been attributed to a naturally pyramided complex involving three major genes: one race-nonspecific gene, Vr, conditioning resistance to all known races, plus two race-specific genes. The race-nonspecific gene was identified as an independently segregating gene by Dayton and Williams (1968) and is referred to in this paper as Vr-DW. The first researchers to study the scab resistance gene complex in Russian apple never described the phenotype conditioned by the race-nonspecific gene. Later, Aldwinckle et al. (1976) associated the name Vr with a scab resistance gene conditioning distinctive stellate necrotic reactions, which we refer to as Vr-A in order to distinguish it from Vr-DW. We show that the segregation ratios in progenies from the scab differential hosts 2 and 4 that are derived from Russian apple, crossed with susceptible cultivars were consistent with a single gene conditioning resistance in each host. The genes have been named Vh2 and Vh4, respectively. Resistant segregants from host 2 showed stellate necrotic reactions, while those from host 4 showed hypersensitive reactions. Both the phenotypes and the genetic maps for the genes in the respective hosts were very similar to those of the genes previously named Vr-A and Vx, respectively, in an F1 family of Russian apple. We showed that race 2 of V. inaequalis isolated from host 2 was able to infect resistant descendants of the non-differential accession PRI 442-23 as well as host 2. The descendants of PRI 442-23 were expected to carry the race-nonspecific Vr-DW gene, but in fact carry Vr-A. We conclude that the Vh2 gene in host 2 and Vr-A are the same, and that the Vh4 gene in host 4 and Vx are the same. However, a major finding of this study is that the latter gene mapped to linkage group 2 of apple instead of linkage group 10 as suggested from previous research. With the two race-specific genes from Russian apple defined now, we discuss the nature of the race-nonspecific Vr-DW gene in this accession. We also report the identification of a new scab resistance gene, VT57, from either Golden Delicious or Red Dougherty, which conditions chlorotic resistance reactions and is linked to Vh2.     

Cloning and functional characterization of the Rvi15 (Vr2) gene for apple scab resistance

Apple scab, caused by Venturia inaequalis, is a serious disease of apple. Previously, the scab resistance Rvi15 (Vr2) from the accession GMAL 2473 was genetically mapped, and three candidate resistance genes were identified. Here, we report the cloning and functional characterization of these three genes, named Vr2-A, Vr2-B, and Vr2-C. Each gene was cloned with its native promoter, terminator and introns, and inserted into the susceptible apple cultivar ‘Gala’. Inoculation of the plants containing Vr2-A and Vr2-B induced no resistance symptoms, but abundant sporulation. However, inoculation of the plants harboring Vr2-C showed a hypersensitive response with clear pinpoint pits, and no or very little sporulation. We conclude that Vr2-C is the Rvi15 (Vr2) gene. This gene belongs to the Toll and mammalian interleukin-1 receptor protein nucleotide-binding site leucine-rich repeat structure resistance gene family. The proteins of this gene family reside in the cytoplasm, whereas V. inaequalis develops in the apoplast, between the epidermis and cuticle, without making haustoria. The spatial separation of the recognizing resistance protein and the pathogen is discussed. This is the second cloned gene for apple scab resistance, and out of these two the only one leading to a symplastic protein.     

Genetics of resistance in apple against <Emphasis Type="Italic">Venturia inaequalis</Emphasis> (Wint.) Cke

Apple (Malus × domestica) is the third important fruit in terms of production and consumption worldwide. Apple scab caused by Venturia inaequalis is the most devastating disease of apple. In the apple-growing regions, many fungicides are sprayed to control the disease leading to increase in the production cost. Development of scab-resistant cultivars is the long-lasting solution to control the disease. In apples, more than 20 major scab resistance genes have been identified in various cultivars and few wild relatives. Of all these genes, Rvi6 derived from Malus floribunda has been most extensively used in different breeding programs. Gene for gene interactions of these resistance genes with the avirulence genes from V. inaequalis have been understood in many cases. QTL-based polygenic resistance has also been characterized in apple. Nucleotide Binding Site Leucine-Rich Repeats (NBS-LRR) have been identified from the apple genome and many of them have been characterized from the scab resistance region. Molecular markers associated with most of the major scab resistance genes have been identified and their position has been mapped on different linkage groups. Marker-assisted selection (MAS) can be helpful in speeding up and accurately identifying the scab-resistant parents and progeny. Pyramiding of several major resistance genes can be undertaken for more durable resistance against apple scab. The present paper reviews the Malus-Venturia pathosystem, current status of knowledge about scab resistance genes, and their application in breeding against apple scab.