To determine host resistance to plant parasite P. aegyptiaca, we used a pot system containing soil infested with P. aegyptiaca seeds. In the current study, concomitant with the decrease of SL content, there were significant decreases in the number of parasite tubercles and shoots growing on tomato lines 1, 2, 5 and 11 relative to the control treatment was observed. Line 5 exhibited the highest resistance to P. aegyptiaca infestation. In contrast, the non-mutated wild-type plants were highly susceptible to the parasite infestation.To date, several unique SLs, including orobanchol and solanacol, have been reported in the root exudates and extracts of tomato . Since orobanchol is a major SL in tomato root exudates, and is a specific germination stimulant for P. aegyptiaca, we determined orobanchol content in the roots of CCD8Cas9 mutant lines. Orobanchol levels were found to be significantly decreased in the CCD8Cas9 mutated lines relative to the wild type. Interestingly, orobanchol was not detectable in line 5, which was shown to be the most resistant to P. aegyptiaca infestation. The higher orobanchol content and only partial resistance to P. aegyptiaca obtained in line 11a could be explained by either the deletion of the cyclic amino acid Pro-244 which is involved in the protein’s structural and conformational rigidity, indoor weed growing accessories can partially restore protein functionality or imbalanced carotenoid profile still provide support in line 11 to produce SL.
However, in line 2a, the deletion of 1 amino acid strongly affected CCD8 activity, as reffected by significant decreases in orobanchol content and P. aegyptiaca infestation. Our study also reports that CCD8Cas9 mutated lines have increased transcript levels of PDS1, LCY-β and altered carotenoid contents. This could be due to the decrease in SL content positively affecting the feedback regulation of these upstream biosynthetic-pathway genes. Another study showed that when the carotenoid-bio-synthesis pathway gene PDS1 is inhibited by furidone treatment, tomato seedlings show a strong decrease in SL content and less germination of P. ramosa compared to the wild-type treatment. These results indicate that inhibition of the carotenoid-biosynthesis pathway not only affects carotenoid biosynthesis but is also involved in modulation of downstream SL pathways and vice versa. Hence, there is a tight correlation between SL production, carotenoid biosynthesis and the resistance mechanism. Previous studies demonstrated that SL-biosynthesis pathways are strictly controlled through negative-feedback inhibition such as D10 transcript levels are elevated in the enhanced-branching d10-1 dwarf mutants of rice and RMS1 transcript level increased dramatically in ramosus mutants of pea and treatment with SL analog GR24 leads to restore the transcripts level.
Based on these results, we expected changes in the expression of CCD8 transcripts in our mutants. To measure CCD8 expression, we analyzed its relative transcript abundance using quantitative real-time PCR. Interestingly, the CCD8 transcript level was substantially increased in the transgenic CCD8Cas9 mutated tomato plants relative to the wild-type plants . In addition to decrease in orobanchol content, line 5 had shown highly increase in the transcript level in comparison to line 1, 2 and 11, which are negatively regulated by SL, provide a basis to explain its higher resistance against P. aegyptiaca compared to other lines. Similar results have been also reported by others, where expression of the SL-biosynthesis gene in Arabidopsis was upregulated in SL-biosynthesis-deffective mutants , suggesting that SL content influences the transcript level of genes involved in SL biosynthesis. Another study found increased transcript level of SlD27 and SlCCD8 in tomato roots after P. ramosa infection, suggesting activation of the SL-biosynthesis pathway upon interaction of plant parasite with host root. In our previous studies, we showed that production of siRNA molecules in tomato targeting P. aegyptiaca genes facilitates suppression of the parasite genes and results in enhanced resistance to the parasite. Intriguingly, in a recent study, SL-deficient tomato lines displayed increased tubercle development and enhanced infestation with pre-germinated P. ramosa seeds. We assume that the contrast with our results is due to difference in mechanism of germination and susceptibility.
In contrast to previous study, we do not pre germinate the parasite seeds, we directly triggered SL-deficient tomato lines with the parasite P. aegyptiaca seeds because P. aegyptiaca seeds become SL independent upon pre-germination. Another possible explanation for the increased parasitic infection in SlCCD8-RNAi lines, might be their enhanced auxin transport or altered auxin levels, as has been reported for the Arabidopsis SL-deficient mutant max4. In this study, CRISPR/Cas9 was used to precisely knock out the CCD8 gene in tomato. Disease resistance based on the editing of specific genes can be easily applied to other susceptible crops without long-term back crossing, and even those that are distantly related. This method is expected to be effective against other Phelipanche and Orobanche species, if the parasite species share sufficient homology in their target sequences. In addition, the mutated plants are devoid of foreign DNA sequences and are not considered genetically modified organisms. One of the significant limitations of the CRISPR/Cas9 system is the generation of significant of-target cleavage as a result of non-specificity of the sgRNA with mismatched complementary target DNA within the genome. Thus, that may alter the function of a gene and may result in genomic instability. Although, several modifications of the Cas9 enzyme and sgRNA designing methods have been developed to increase target specificity, the of-target cleavage is still considered as major limiting factor with CRISPR/Cas9 technology. Additionally, under regulatory regimes in many countries, the new breeding technology CRISPR/Cas9 does not fall under the definition of a GMO, but in the EU countries CRISPR/Cas9 is still considered as GMO. In conclusion, using CRISPR/Cas9, we generated an effective mutation in tomato to be executed in breeding programs for resistance against parasitic weeds. In the current study, we demonstrate that genetic resistance to parasitic weeds can be created using CRISPR/Cas9. We used this technology to edit CCD8 in tomato. We chose to knock out the CCD8 gene due to its role in SL production and its clear morphological phenotype . We realize that mutations in CCD8 can negatively affect tomato plant morphology; however, to avoid this issue, our system could be used in a rootstock grafed to a wild-type scion, and might be combined with tomato rootstocks that are already resistant to fungal pathogens, viruses, and nematodes. Further study is needed with grafing experiments to investigate this concept, which might be applicable to a wide range of crop plants. To the best of our knowledge, this study is the first to report the generation of tomato plants that are resistant to the weedy parasite P. aegyptiaca using the CRISPR/ Cas9 genome-editing technology.The weed control data showed no significant interaction between sites. The rice response data, however, showed significant treatment by site interaction for bleaching symptoms but not for chlorosis, stunting, stand reduction, necrosis symptoms ratings, and yield. Therefore, vertical grow rack system the data for bleaching were analyzed separately by site while weed control data and all other injury symptoms and yield for both sites were combined. Weed population composition varied at the two sites for this experiment. Site one had control plots dominated by sedges and broadleaf weeds, averaging 73% coverage, while Echinochloa spp. abundance averaging 2% and bearded sprangletop abundance averaging 5% based on the treated control plots . The control plots at site two had no sedge or broadleaf weeds and was dominated by Echinochloa spp. and bearded sprangletop averaging 32 and 15%, respectively . Weed populations could differ at these two sites because of differences in management practices, flooding time, and planting dates. All treatments, including the grower standard, across both sites provided complete season long Echinochloa spp. control . The grower standard treatment had 100% control of bearded sprangletop early in the season, then decreased to 99% control by the end of rice heading stage. Every treatment of tetflupyrolimet had season long complete control of bearded sprangletop. All treatments were statistically similar for control of bearded sprangletop and no PRE herbicide applications showed escapes as described in other experiments due to its delayed emergence .
Tetflupyrolimet’s season long control of these problem grass weed species, both at the PRE and POST timings, may be useful for growers to rotate a new MOA in their herbicide program. This herbicide provides an opportunity to eliminate part of the populations of these problem grass weeds that have resistance as outlined in a field survey of California rice weeds by Becerra-Alvarez et al. in 2022 and 2023. Becerra-Alvarez also tested tetflupyrolimet against all submitted grass samples, finding complete control in all samples . Rice injury symptoms observed were slight bleaching, chlorosis, stunting, and stand reduction across both sites. At both sites, moderate early bleaching was observed in the grower standard treatments and then subsided later in the season . This is not surprising due to the extensive characterization of rice’s response to the bleaching herbicide, clomazone . The grower standard treatment also showed moderate stunting at 28 DAT due to the early POST application of penoxsulam, which is known to cause stunting and chlorosis in rice . PRE and POST tetflupyrolimet treatments did not show any bleaching throughout the season. Chlorosis, stunting, and stand reduction symptoms in all tetflupyrolimet treatments averaged across both sites were overall minimum, ranging from 0 to 8% by 28 DAT . All symptoms observed were slight and the rice fully recovered by 42 DAT . There was no significant interaction between treatments for any of these symptoms and no trend of increased symptoms with increased rate or varied application timing. These slight symptoms were not surprising and were possibly due to the cooler nights recorded around Biggs, CA in 2023, averaging 14 C during the germination and early vegetative stages . Yield for the grower standard treatment averaged 5,385 kg ha-1 . Yields of tetflupyrolimet treatments were not significantly different than grower standard treatments and ranged between 5,768 to 6,295 kg ha-1 , which indicated the slight early injury observed in tetflupyrolimet treatments did not affect yield. As discussed previously, the 2023 growing season experienced colder than usual temperatures, which could be a contributing factor of the lower yield measured in this study . Although temperature alone could not account for this experiment’s low yields, a late planting date and rice lodging after heading also may have contributed to the low yields .There was significant treatment by year interaction for weed control, so the data were analyzed separately by year for Echinochloa spp., bearded sprangletop, ricefield bulrush, small flower umbrellas edge, duck salad, and redstem. There was also significant treatment by year interaction for yield, while no significant interaction was detected for rice chlorosis, bleaching, stunting, stand reduction, and necrosis. Therefore, all weed control and yield data were analyzed separately by year while all rice injury symptom data were combined. Weed population composition varied each year: in 2022, there was a lower density of sedge and broad leaves than in 2023; however, the dominant species for both years was Echinochloa spp. In 2022, the untreated control plots had a high abundance of Echinochloa spp. and bearded sprangletop cover averaging 62% of the plot, while the sedges and broad leaves had an average of 23% relative cover by 4 LSR . In 2023, the untreated controls plots had a higher abundance of both grasses, averaging 74%, as well as sedges and broad leaves, averaging 45% abundance . This shift in weed population composition is not surprising and has been observed before in previous experiments near this site due to differences in soil moisture and temperature throughout the growing season . In 2022, the grower standard treatment showed 94% control of Echinochloa spp. at 94% throughout the season . This was significantly lower than all treatments that included tetflupyrolimet, which had 98 to 100% control of Echinochloa spp. ranging from 98 to 100% throughout the season. The grower standard treatment showed complete control of bearded sprangletop at 14 DAT, but minimally decreased to 99% control by 56 DAT. The PRE tetflupyrolimet treatments showed a similar trend with bearded sprangletop control, shifting from complete control at 14 DAT to decreasing by a few percent ranging from 97 to 100%. However, POST applications of tetflupyrolimet, which were in combination with a PRE herbicide that has herbicidal activity on grasses, maintained complete control throughout the season .In 2023, the grower standard treatment maintained complete control of Echinochloa spp. throughout the entire season .