Trans-specific gene silencing of acetyl-CoA carboxylase in a root-parasitic plant.

Parasitic species of the family Orobanchaceae are devastating agricultural pests in many parts of the world. The control of weedy Orobanchaceae spp. is challenging, particularly due to the highly coordinated life cycles of the parasite and host plants. Although host genetic resistance often provides the foundation of plant pathogen management, few genes that confer resistance to root parasites have been identified and incorporated into crop species. Members of the family Orobanchaceae acquire water, nutrients, macromolecules, and oligonucleotides from host plants through haustoria that connect parasite and host plant roots. We are evaluating a resistance strategy based on using interfering RNA (RNAi) that is made in the host but inhibitory in the parasite as a parasite-derived oligonucleotide toxin. Sequences from the cytosolic acetyl-CoA carboxylase (ACCase) gene from Triphysaria versicolor were cloned in hairpin conformation and introduced into Medicago truncatula roots by Agrobacterium rhizogenes transformation. Transgenic roots were recovered for four of five ACCase constructions and infected with T. versicolor against parasitic weeds. In all cases, Triphysaria root viability was reduced up to 80% when parasitizing a host root bearing the hairpin ACCase. Triphysaria root growth was recovered by exogenous application of malonate. Reverse-transcriptase polymerase chain reaction (RT-PCR) showed that ACCase transcript levels were dramatically decreased in Triphysaria spp. parasitizing transgenic Medicago roots. Northern blot analysis identified a 21-nucleotide, ACCase-specific RNA in transgenic M. truncatula and in T. versicolor attached to them. One hairpin ACCase construction was lethal to Medicago spp. unless grown in media supplemented with malonate. Quantitative RT-PCR showed that the Medicago ACCase was inhibited by the Triphysaria ACCase RNAi. This work shows that ACCase is an effective target for inactivation in parasitic plants by trans-specific gene silencing.

Characterization of an endophytic bacterium (Pseudomonas aeruginosa), originating from tomato (Solanum lycopersicum L.), and its ability to inhabit the parasitic weed Phelipanche aegyptiaca.

PHELIPANCHE AEGYPTIACA: is an obligate holo-parasitic weedlacking a functional photosynthetic system, which subsists on roots of a wide range of host crops, causing severe losses in yield quality and quantity. The parasite and its host are connected through their vascular system, forming a unique ecological system that enables the exchange of various substances. In a previous study, it was suggested that endophytic bacteria, which naturally inhabit the internal tissues of plants, can also be transmitted from the parasitic weed to its host and vice versa. In the current study, we investigate the characteristics of a previously isolated Pseudomonas sp. PhelS10 strain using both biochemical and molecular methods. This isolate was obtained from tomato plant tissue and was able to reduce P. aegyptiaca parasitism, and thus it may serve as a biocontrol agent. Our results revealed that production of Pseudomonas aeruginosa quinolone signal (PQS) was 2.1 times higher than that of the standard Pseudomonas aeruginosa strain (PAO1), which contributed to a 22% higher biofilm formation capability. PhelS10 strain was detected in the xylem of tomato plants using FISH analysis. In addition, PhelS10 strain was found in the parasitic weed's inner tissues, confirming the hypothesis that endophytic bacteria traffic between the host plant and its parasitic weed.

Susceptibility of Phelipanche and Orobanche species to AAL-toxin.

Fusarium and Alternaria spp. are phytopathogenic fungi which are known to be virulent on broomrapes and to produce sphinganine-analog mycotoxins (SAMs). AAL-toxin is a SAM produced by Alternaria alternata which causes the inhibition of sphinganine N-acyltransferase, a key enzyme in sphingolipid biosynthesis, leading to accumulation of sphingoid bases. These long chain bases (LCBs) are determinant in the occurrence of programmed cell death (PCD) in susceptible plants. We showed that broomrapes are sensitive to AAL-toxin, which is not common plant behavior, and that AAL-toxin triggers cell death at the apex of the radicle as well as LCB accumulation and DNA laddering. We also demonstrated that three Lag1 homologs, encoding components of sphinganine N-acyltransferase in yeast, are present in the Orobanche cumana genome and two of them are mutated leading to an enhanced susceptibility to AAL-toxin. We therefore propose a model for the molecular mechanism governing broomrape susceptibility to the fungus Alternaria alternata.

Agrobacterium rhizogenes-mediated transformation of the parasitic plant Phtheirospermum japonicum.

BACKGROUND: Plants within the Orobanchaceae are an agriculturally important group of parasites that attack economically important crops to obtain water and nutrients from their hosts. Despite their agricultural importance, molecular mechanisms of the parasitism are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We developed transient and stable transformation systems for Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae. The transformation protocol was established by a combination of sonication and acetosyringone treatments using the hairy-root-inducing bacterium, Agrobacterium rhizogenes and young seedlings. Transgenic hairy roots of P. japonicum were obtained from cotyledons 2 to 3 weeks after A. rhizogenes inoculation. The presence and the expression of transgenes in P. japonicum were verified by genomic PCR, Southern blot and RT-PCR methods. Transgenic roots derived from A. rhizogenes-mediated transformation were able to develop haustoria on rice and maize roots. Transgenic roots also formed apparently competent haustoria in response to 2,6-dimethoxy-1,4-benzoquinone (DMBQ), a haustorium-inducing chemical. Using this system, we introduced a reporter gene with a Cyclin B1 promoter into P. japonicum, and visualized cell division during haustorium formation. CONCLUSIONS: We provide an easy and efficient method for hairy-root transformation of P. japonicum. Transgenic marker analysis revealed that cell divisions during haustorium development occur 24 h after DMBQ treatment. The protocols described here will allow functional analysis of genes involved in plant parasitism.