Bioscience Methods 2026, Vol.17, No.1, 9-22 http://bioscipublisher.com/index.php/bm 15 without cutting DNA (Ding et al., 2022; Clark et al., 2024). CRISPRa can also be timed, for example after flowering or during fruit growth, to protect fruit from black rot when needed. This method has worked in Arabidopsis and may also be applied to pineapple. Overall, increasing the strength of existing defenses by promoter edits or CRISPRa is a practical approach. It uses the plant’s own genes, works precisely, and can avoid big growth penalties. 4.2 Knockout of susceptibility genes to block pathogen entry A basic CRISPR way to add disease resistance is to delete susceptibility (S) genes. These are plant genes that pathogens use to get in. When these genes stop working, plants may gain resistance (Wan et al., 2020; Moffa et al., 2025). One prime candidate is pineapple’s ortholog of MLO. Powdery mildew is not a known pineapple disease (likely due to environmental differences), but MLOgenes also can influence other pathogens or developmental processes. If pineapple MLO contributes to any fungal infection (perhaps leaf spot fungi), knocking it out might be beneficial. More directly, consider S genes for bacterial pathogens: many bacterial diseases require host susceptibility factors for infection. In citrus, CsLOB1 was an S gene for canker that CRISPR was used to disable (Jia and Wang, 2020). Pineapple’s bacterial heart rot (caused by Dickeyaor related Erwinia) may similarly hinge on a host factor. Research in related bromeliads or monocots could point to such factors – for instance, Erwinia chrysanthemi (which causes soft rot in many plants) often secretes pectate lyases and cell wall-degrading enzymes; plants with altered cell wall components can show resistance. A hypothetical S gene might be a pineapple pectin acetylesterase gene required for the bacterium to break down pectin. If so, a CRISPR knockout of that gene could reduce tissue maceration and confer partial resistance to heart rot. Another category of S genes are those involved in negative immune regulation. For example, EDR1 is a kinase acting as a negative regulator of defense; wheat plants edited for all three homeologs of TaEDR1 showed enhanced broad resistance to powdery mildew (Zhang et al., 2017; Zhou et al., 2023). Pineapple has its own EDR1 homolog – knocking it out via CRISPR might hyperactivate the plant’s basal defenses, making it less susceptible to a range of pathogens (albeit with vigilance for any trade-offs like stunted growth). Given pineapple’s relatively small R gene arsenal, targeting S genes is attractive because it removes the weak links that pathogens universally rely on. CRISPR’s precision is key: undesirable alleles can be cleanly knocked out without disrupting other genes. In tomato, CRISPR deletion of a few amino acids in the SlMlo1 gene conferred mildew resistance and was accomplished within 10 months, producing a variety termed “Tomelo” (Nekrasov et al., 2017; Schenke and Cai, 2020). Pineapple’s longer generation time means the process would be slower, but still far faster than conventional breeding and without altering fruit traits. Importantly, resistance via S gene loss is often broad-spectrum and durable because the pathogen cannot easily overcome the absence of a host factor (as opposed to single R gene resistance which pathogens can defeat by mutating an effector). Hence, creating pineapple knockouts for key S genes – once identified – could yield varieties with stable resistance to heart rot, black rot, or other diseases. The challenge is to pinpoint the right targets: candidate gene discovery might involve transcriptomics (seeing which host genes are upregulated by infection) or ortholog knowledge from other crops. Nonetheless, the concept is clear: if the pathogen needs a host gene, take that gene away using CRISPR, and the plant becomes an unwelcome host. 4.3 Editing regulatory networks: targeting defense regulators and microRNAs CRISPR can do more than change single genes. It can also adjust pineapple’s immune control system to react faster or stay stronger against pathogens. This means editing key regulator genes, such as transcription factors or microRNAs, that control many resistance genes at once.
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