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.

Summary: Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6‐phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277‐bp fragment from Orobanche aegyptiaca M6PR‐mRNA, in an inverted‐repeat configuration. M6PR‐siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT‐PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%–80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene‐silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.

Background: The yeast Metschnikowia fructicola is an antagonist with biological control activity against postharvest diseases of several fruits. We performed a transcriptome analysis, using RNA-Seq technology, to examine the response of M. fructicola with citrus fruit and with the postharvest pathogen, Penicillium digitatum. Results: More than 26 million sequencing reads were assembled into 9,674 unigenes. Approximately 50% of the unigenes could be annotated based on homology matches in the NCBI database. Based on homology, sequences were annotated with a gene description, gene ontology (GO term), and clustered into functional groups. An analysis of differential expression when the yeast was interacting with the fruit vs. the pathogen revealed more than 250 genes with specific expression responses. In the antagonist-pathogen interaction, genes related to transmembrane, multidrug transport and to amino acid metabolism were induced. In the antagonist-fruit interaction, expression of genes involved in oxidative stress, iron homeostasis, zinc homeostasis, and lipid metabolism were induced. Patterns of gene expression in the two interactions were examined at the individual transcript level by quantitative real-time PCR analysis (RT-qPCR). Conclusion: This study provides new insight into the biology of the tritrophic interactions that occur in a biocontrol system such as the use of the yeast, M. fructicola for the control of green mold on citrus caused by P. digitatum.

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.