Broomrapes (Phelipanche aegyptiaca and Orobanche spp.) are obligate plant parasites that cause extreme damage to crop plants. The parasite seeds have strict requirements for germination, involving preconditioning and exposure to specific chemicals strigolactones [SLs] exuded by the host roots. SLs are plant hormones derived from plant carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase 8 (CCD8). Having no effective means to control parasitic weeds in most crops, and with CRISPR/Cas9 being an effective gene-editing tool, here we demonstrate that CRISPR/Cas9-mediated mutagenesis of the CCD8 gene can be used to develop host resistance to the parasitic weed P. aegyptiaca. Cas9/single guide (sg) RNA constructs were targeted to the second exon of CCD8 in tomato (Solanum lycopersicum L.) plants. Several ^CCD8Cas9 mutated tomato lines with variable insertions or deletions in CCD8 were obtained with no identified off-targets. Genotype analysis of T1 plants showed that the introduced CCD8 mutations are inherited. Compared to control tomato plants, the ^CCD8Cas9 mutant had morphological changes that included dwarfing, excessive shoot branching and adventitious root formation. In addition, SL-deficient ^CCD8Cas9 mutants showed a significant reduction in parasite infestation compared to non-mutated tomato plants. In the ^CCD8Cas9 mutated lines, orobanchol (SL) content was significantly reduced but total carotenoids level and expression of genes related to carotenoid biosynthesis were increased, as compared to control plants. Taking into account, the impact of plant parasitic weeds on agriculture and difficulty to constitute efficient control methods, the current study offers insights into the development of a new, efficient method that could be combined with various collections of resistant tomato rootstocks.

Deletants of the sphingolipid biosynthetic pathway genes FEN1 and SUR4 of Saccharomyces cerevisiae , as well as deletants of their orthologs in Candida albicans , were found to be 2- to 5-fold-more sensitive to amphotericin B (AmB) than parent strains. The inhibition of sphingolipid biosynthesis in parent strains by myriocin sensitized them to AmB, which can be reversed by providing phytosphingosine, an intermediate in the sphingolipid pathway. These results indicate that sphingolipids modulate AmB resistance, with implications for mechanisms underlying AmB action and resistance.

Invasive opportunistic fungal infections of humans are common among those sufferingfrom impaired immunity and are difficult to treat resulting in high mortality.Amphotericin B (AmB) is one of the few antifungals available to treat suchinfections. The AmB resistance mechanisms reported so far mainly involve decrease inergosterol content or alterations in cell wall. In contrast, depletion ofsphingolipids sensitizes cells to AmB. Recently, overexpression of PMP3 gene,encoding plasma membrane proteolipid 3 protein, was shown to increase and itsdeletion to decrease, AmB resistance. Here we have explored the mechanistic basis ofPMP3 effect on AmB resistance. It was found that ergosterol content andcell wall integrity are not related to modulation of AmB resistance by PMP3.A few prominent phenotypes of PMP3 delete strain, namely, defective actinpolarity, impaired salt tolerance and reduced rate of endocytosis are also notrelated to its AmB-sensitivity. However, PMP3 overexpression mediatedincrease in AmB resistance requires a functional sphingolipid pathway. Moreover, AmBsensitivity of strains deleted in PMP3 can be suppressed by the addition ofphytosphingosine, a sphingolipid pathway intermediate, confirming the importance ofthis pathway in modulation of AmB resistance by PMP3.

Root parasitic weeds infect numerous economically important crops, affecting total yield quantity and quality. A lack of an efficient control method limits our ability to manage newly developing and more virulent races of root parasitic weeds. To control the parasite induced damage in most host crops, an innovative biotechnological approach is urgently required. Strigolactones (SLs) are plant hormones derived from carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase (CCD) 7, CCD8 and More Axillary Growth 1 (MAX1) genes. SLs act as branching inhibitory hormones and strictly required for the germination of root parasitic weeds. Here, we demonstrate that CRISPR/Cas9-mediated targted editing of SL biosynthetic gene MAX1, in tomato confers resistance against root parasitic weed Phelipanche aegyptiaca. We designed sgRNA to target the third exon of MAX1 in tomato plants using the CRISPR/Cas9 system. The T₀ plants were edited very efficiently at the MAX1 target site without any non-specific off-target effects. Genotype analysis of T₁ plants revealed that the introduced mutations were stably passed on to the next generation. Notably, MAX1-Cas9 heterozygous and homozygous T₁ plants had similar morphological changes that include excessive growth of axillary bud, reduced plant height and adventitious root formation relative to wild type. Our results demonstrated that, MAX1-Cas9 mutant lines exhibit resistance against root parasitic weed P. aegyptiaca due to reduced SL (orobanchol) level. Moreover, the expression of carotenoid biosynthetic pathway gene PDS1 and total carotenoid level was altered, as compared to wild type plants. Taking into consideration, the impact of root parasitic weeds on the agricultural economy and the obstacle to prevent and eradicate them, the current study provides new aspects into the development of an efficient control method that could be used to avoid germination of root parasitic weeds.