The Phtheirospermum japonicum isopentenyltransferase PjIPT1a regulates host cytokinin responses in Arabidopsis

The hemiparasitic plant Phtheirospermum japonicum is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyse the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter-reporter, and further analysed the subcellular localization, the enzymatic function, and contribution to inducing hypertrophy by studying CRISPR-Cas9 induced Phtheirospermum mutants. PjIPT1a was specifically expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed isopentenyltransferases activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a with shared characteristics was also identified in the related weed Striga hermonthica. With PjIPT1a we identified a bona fide parasitism gene. We propose that PjIPT1a exemplifies how parasitism-related functions evolve through gene duplications and neofunctionalization.

Broad-spectrum Resistance to Root-Knot Nematodes in Transgenic Cucurbits

Root-knot nematodes (RKN), Meloidogyne spp., are extremely destructive pathogens of cucurbit crops grown in the United States and Israel. The safety and environmental concerns of toxic nematicides, and limited sources of natural cucurbit resistance to the four major species of Meloidogyne that threaten these crops in Israel and the U.S., have emphasized the use of biotechnology to develop cucurbits with novel RKN resistance. The U.S. scientists have identified over 40 unique RKN parasitism genes that encode nematode secretions involved in successful plant root infection by RKN, and they have demonstrated that expression of a double-stranded RNA (dsRNA) complementary to a RKN parasitism gene (called 16DIO) in Arabidopsis thaliana induced RNA interference (RNAi)-mediated silencing of the RKN16DlO gene and produced transgenic plants with strong resistance to all four major RKN species. The expression 8D05 parasitism gene was found to coincide with the timing of upregulation of NtCel7 promoter (identified to be upregulated in giantcells by US scientists). NtCel7 promoter was used to express the genes at the right time (early stages of infection) and in the right place (giant-cells) in transgenic plants. US partners produced NtCel7 (nematode-induced promoter)-driven 16DlO-RNAi and 8DOS-RNAi constructs, pHANNIBAL 4D03-RNAi construct and modified 16DlO-RNAi construct (for increased RNAi expression and efficacy) for cucurbit transformation in Israel. In Arabidopsis, some 16DlO-RNAi plant lines show greater levels of resistance to M. incognita than others, and within these lines resistance of greater than 90% reduction in infection is observed among almost all replicates in US. The level of observed nematode resistance is likely to be directly correlated with the level of RNAi expression in individual plants. In Israel, all the RKN parasitism genes-RNAi constructs were successfully transformed into cucumber and melon. The transgenic lines were evaluated for expression of the transgene siRNA in leaves and roots. Those displaying transgene siRNA accumulation were passed on for nematode resistance analysis. Rl seedlings from different lines were subjected to evaluation for resistance to M. javanica. None of the lines was resistant to the nematode in contrast with US partner's results in Arabidopsis. This could be for the following reasons: a) The level of transgene siRNA was insufficient in cucumber and tomato to cause resislance. b) 111e nemalode species on cucwnber IIlay be different ur act in a different manner. c) The assay was performed in soil with a high level of nematode inoculation, and not in petri dish, which may not permit the observation of a low level of resistance.

The 8D05 Parasitism Gene of Meloidogyne incognita Is Required for Successful Infection of Host Roots

Parasitism genes encode effector proteins that are secreted through the stylet of root-knot nematodes to dramatically modify selected plant cells into giant-cells for feeding. The Mi8D05 parasitism gene previously identified was confirmed to encode a novel protein of 382 amino acids that had only one database homolog identified on contig 2374 within the Meloidogyne hapla genome. Mi8D05 expression peaked in M. incognita parasitic second-stage juveniles within host roots and its encoded protein was limited to the subventral esophageal gland cells that produce proteins secreted from the stylet. Constitutive expression of Mi8D05 in transformed Arabidopsis thaliana plants induced accelerated shoot growth and early flowering but had no visible effects on root growth. Independent lines of transgenic Arabidopsis that expressed a double-stranded RNA complementary to Mi8D05 in host-derived RNA interference (RNAi) tests had up to 90% reduction in infection by M. incognita compared with wild-type control plants, suggesting that Mi8D05 plays a critical role in parasitism by the root-knot nematode. Yeast two-hybrid experiments confirmed the specific interaction of the Mi8D05 protein with plant aquaporin tonoplast intrinsic protein 2 (TIP2) and provided evidence that the Mi8D05 effector may help regulate solute and water transport within giant-cells to promote the parasitic interaction.