Powdery mildews (PM) are common and severe pathogen groups that threaten plants, and PM resistance is complex and polygenic in cucumbers. Previously mlo-based resistance was reported in various plants, including cucumber, with generated loss-of CsaMLO function mutants. However, mlo-based resistance in cucumber is also complex and involves additional mechanisms such as hypersensitive response (HR) and papillae formation. For this reason, we focused on determining the mlo-based powdery mildew resistance mechanism in cucumber. CRISPR/Cas9 was used in the present study to generate loss-of-function mutants for CsaMLO1, CsaMLO8, and CsaMLO11 of PM susceptible ADR27 cucumber inbred lines and CsaMLO mutants were obtained and validated. Trypan Blue and DAB staining were performed to detect Podosphaera xanthii germination/penetration rates and accumulation of Reactive Oxygen Species (ROS). Our results indicate that PM-susceptibility associated CsaMLOs in cucumber are negative regulators in different defense mechanisms against powdery mildew at early and late stages of infection. Further, the experiment results indicated that CsaMLO8 mutation-based resistance was associated with the pre-invasive response, while CsaMLO1 and CsaMLO11 could be negative regulators in the post-invasive defense response in cucumber against P. xanthii. Although the loss-of CsaMLO8 function confers the highest penetration resistance, CsaMLO1 and CsaMLO11 double mutations could be potential candidates for HR-based resistance against PM pathogen in cucumber. These results highlighted the crucial role of CRISPR/Cas9 to develop PM resistant cucumber cultivars, possessing strong pre-invasive defense with CsaMLO8 or post-invasive with CsaMLO1/CsaMLO11 mutations.

CRISPR/Cas9 is one of the most robust technologies for plant breeding enabling precise and efficient modifications in a genome. This technology is being used for the manipulation of target genes in a host to develop resistance against the plant pathogens. Cucumis sativus elF4E is one of the target genes playing a key role in viral infection during interaction with potyvirus viral proteins genome linked (VPg). Nevertheless, the allelic and positional effect of elF4E mutations in C. sativus is to be clarified in elF4E-VPg interaction. In addition, there are entanglements in the massive production of pathogen-resistant cultivars suitable for commercial production using CRISPR/Cas9 technology. Therefore, we targeted different positions of the elF4E in G27 and G247 inbred lines, using specific gRNA1 and gRNA2 for the first and third exons, respectively, and 1,221 transgene-free plants were selected in segregated T1 generation, where 192 G27 and 79 G247 plants had the least mutation at Cas9 cleavage site of gRNA1 or gRNA2. Crossing was performed to see allelic effects of elfF4E mutations in F1 populations, which were homozygous and heterozygous single (elF4E_1DEL or elF4E_3DEL) and double (elF4E_1-3DEL) mutants. Disease symptoms of watermelon mosaic virus (WMV), papaya ringspot virus (PRSV), and zucchini yellow mosaic virus (ZYMV) were evaluated in both non-edited and edited F1 plants, and we did not observe any symptom in homozygous elF4E_1-3DEL and elF4E_1DEL mutants. However, homozygous elF4E_3DEL was positive in reverse transcription polymerase chain reaction (RT-PCR), even if there were no significant symptoms on the inoculated leaves. ELISA and qRT-PCR indicated lower viral accumulation in homozygous elF4E_3DEL than heterozygous and non-edited plants. Regeneration and transformation protocols were also optimized comprehensively for both the genotypes. The average number of shoots/100 explants was determined for both G27 and G247 as 13.6 and 18.0, respectively. We could not detect any distinguishing difference between the non-edited and edited F1 plants for yield and morphology. Our results demonstrate an effective route for mass production of viral resistant cultivars of cucumber to WMV, ZYMV, and PRSV. In this way, the pathogen-resistant cultivars could be generated to reduce the losses caused by these pathogens in cucumber production.

Podosphaera xanthii causes powdery mildew of cucumber, and is associated with significant yield and quality losses. Development of resistant or tolerant varieties is the most effective and eco-friendly strategy for powdery mildew management. An important host resistance mechanism is based on the recognition of conserved resistance genes, resulting in durable resistance. To determine powdery mildew resistance mechanisms in cucumber, total RNAs were isolated from the powdery mildew resistant cultivar Meltem, the tolerant line VT18, and the susceptible local variety Camlica. Expression levels of nine genes in these plants were analysed by Reverse Transcription Polymerase Chain Reaction (RT-PCR). The host reactions were assessed using microscope observations of stained specimens. Serine/threonine (STN7), transcription factor (WRKY22), serine/threonine-protein kinase (D6PKL1), and serine/threonine receptor kinase (NFP) genes were induced, as positive regulators in defence mechanisms against powdery mildew. Polygalacturonase Inhibitor (PGIP) did not express after P. xanthii inoculation of Camlica, resulting in susceptibility. After inoculation, callose synthase (CALLOSE) and cinnamyl alcohol dehydrogenase (CAD) gene expression levels were increased in resistant Meltem, but Hypersensitive Reaction (HR) and ROS formation were only linked in the tolerant VT18. Powdery mildew development was less in Meltem than in VT18, indicating that cell wall thickening and HR play separate roles in resistance to this disease.

CRISPR drive is a recent and robust tool that allows durable genetic manipulation of the pest population like human disease vectors such as malaria vector mosquitos. In recent years, it has been suggested that CRISPR drives can also be used to control plant diseases, pests, and weeds. However, using a CRISPR drive in Arabidopsis for the first time in 2021 has been shown to use this technology in plant breeding to obtain homozygous parental lines. This perspective has proposed using CRISPR drive to develop pathogen-resistant cultivars by disrupting the susceptibility gene (S). In the breeding program, CRISPR is used to create S-gene mutations in two parental lines of hybrid cultivars. However, CRISPR must be reapplied or long-term backcrossed for the parental line to obtain homozygous S-mutant cultivars. When a parental line crosses with different parental lines to develop new hybrids, heterozygous S-mutations could not resist in hybrid against the pathogen. CRISPR drives are theoretically valid to develop homozygous S-mutant plants against pathogens by only routine pollination after CRISPR drive transformation to just one parental line. This way, breeders could use this parental line in different crossing combinations without reapplying the genome-editing technique or backcrossing. Moreover, CRISPR drive also could allow the development of marker-free resistant cultivars with modifications on the drive cassette.

Kabakgiller Türkiye'de ve dünyada yoğun üretimi yapılan bir sebze grubudur. Külleme hastalıkları, kabakgil üretiminde ekonomik kayıplara neden olan hastalıkların başında gelmektedir. Günümüzde kabakgil külleme etmenleri; Podosphaera xanthii ve Golovinomyces cichoracearum türlerine karşı dayanıklı çeşitler konusunda yapılmış çalışmalar bulunmamaktadır. Bu çalışmanın amacı, Batı Akdeniz ile Doğu Akdeniz arasında yetişen yerel, yabani ve ticari su kabağı, acur, kavun, karpuz, hıyar, sakız kabağı, bal kabağı gibi çeşitli kabakgillerin genetik dayanıklılığını ortaya koymaktır. Çalışmada kabakgillerde verim kaybına neden olan, külleme hastalık etmenleri Podosphaera xanthii ve Golovinomyces cichoracearum türleri hassas hıyar çeşidi (Baccara) üzerinde kültüre alınmıştır. Kültüre alınan külleme etmeni üzerinde yapılan morfolojik, mikroskobik ve moleküler çalışmalar sonucunda patojen Podosphaera xanthii olarak tanımlanmıştır. Hastalık etmeni P. xanthi ile inokule edilen toplam 34 yerel, yabani ve ticari kabakgil çeşidi inokulasyondan sonraki ilk 3 gün boyunca mikroskopta trypan blue, diamino benzidine ve 3,3'-dihexyloxacarbocynin iodide (DiOC6) boyama yöntemleriyle incelenmiştir. Sonraki 7., 14. ve 21. günlerde bitkiler üzerindeki hastalık gelişimleri skorlanarak dayanıklı ve hassas kabakgiller bulunmuştur. Yapılan patojeniste testleri sonucunda VT18, Meltem F1, Poyraz F1 ve 348 ticari hıyar çeşitleri ile Adana kabak, Kaledran hıyar 1 ve Kaledran hıyar 2 yerel çeşitleri en dayanıklı kabakgiller olarak bulunmuştur. Çalışmada Kaledran kavun 2 yerel çeşidi P. xanthii'ye karşı en hassas bitki olarak belirlenmiştir. Çalışmalarda, külleme etmeni P. xanthii'ye karşı dayanıklı olarak bulunan yerel, yabani ve ticari genotipler gelecekteki ıslah çalışmaları için dayanıklılık kaynağı olarak kullanılabilecektir.