Effective RNAi-Mediated Silencing of the Mismatch Repair MSH2 Gene Induces Sterility of Tomato Plants but Not an Increase in Meiotic Recombination

In plant breeding, the ability to manipulate meiotic recombination aids in the efficient construction of new allelic compositions of chromosomes and facilitates gene transfer from wild relatives of crop plants. The DNA mismatch repair system antagonizes meiotic recombination. In this research, a trial was conducted to evaluate transgenic tomato plants carrying an RNA interference (RNAi) construct designed to inhibit the expression of the mismatch repair MSH2 gene. To drive the RNAi construct, we used either a pro-SmAMP2 promoter from Stellaria media ANTIMICROBIAL PEPTIDE2 or a Cauliflower mosaic virus 35S promoter (CaMV35S). The results of real-time PCR showed that, with a 16 h light/8 h dark photoperiod, MSH2-RNAi tomato transgenic plants exhibited MSH2 gene transcript contents ranging from 0% to 3% in the leaves, relative to untransformed controls. However, with this lighting mode, the MSH2-RNAi transgenic plants grew slowly, flowered poorly, and did not form seed sets. During cultivation with a 12 h light/12 h dark photoperiod, MSH2-RNAi transgenic plants exhibited MSH2 gene transcript contents ranging from 3% to 42%, relative to untransformed controls. Under these conditions, F1 hybrid seed sets formed for most of the MSH2-RNAi transgenic plants with the RNAi construct driven by the CaMV35S promoter, and for one transformant with the RNAi construct driven by the pro-SmAMP2 promoter. Under conditions of a 12 h light/12 h dark photoperiod, most of the F1 transgenic hybrids showed MSH2 gene transcript contents ranging from 3% to 34% and formed F2 offspring sets, which made it possible to assess the meiotic recombination frequency. We showed that the effective inhibition of MSH2 in MSH2-RNAi tomato transgenic plants is not associated with an increase in meiotic recombination compared to the control, but it stimulates the sterility of plants. It was established that the expression of the MSH2 gene in tomato plants is about 50 times higher with a 12 h light/12 h dark than with a 16 h light/8 h dark photoperiod. It is discussed that, in Solanum lycopersicum tomato plants, which are not sensitive to the day length for flowering, changing the lighting time may be a means of controlling the meiotic recombination frequency within certain limits.

Application of Double-Strand RNAs Targeting Chitin Synthase, Glucan Synthase, and Protein Kinase Reduces Fusarium graminearum Spreading in Wheat

Controlling the devastating fungal pathogen Fusarium graminearum (Fg) is a challenge due to inadequate resistance in nature. Here, we report on the identification of RNAi molecules and their applications for controlling Fg in wheat through silencing chitin synthase 7 (Chs7), glucan synthase (Gls) and protein kinase C (Pkc). From transgenic Fg strains four RNAi constructs from Chs7 (Chs7RNAi−1, −2, −3, and −4), three RNAi constructs from Gls (GlsRNAi−2, −3, and −6), and one RNAi construct from Pkc (PkcRNAi−5) were identified that displayed effective silencing effects on mycelium growth in medium and pathogenicity in wheat spikes. Transcript levels of Chs7, Gls and Pkc were markedly reduced in those strains. Double-strand RNAs (dsRNAs) of three selected RNAi constructs (Chs7RNAi-4, GlsRNAi-6 and PkcRNA-5) strongly inhibited mycelium growth in vitro. Spray of those dsRNAs on detached wheat leaves significantly reduced lesion sizes; the independent dsRNAs showed comparable effects on lesions with combination of two or three dsRNAs. Expression of three targets Chs7, Gls, and Pkc was substantially down-regulated in Fg-infected wheat leaves. Further application of dsRNAs on wheat spikes in greenhouse significantly reduced infected spikelets. The identified RNAi constructs may be directly used for spray-induced gene silencing and stable expression in plants to control Fusarium pathogens in agriculture.

Synthesis and transfer of RNAi construct for potential knock-down of gene expression of root-knot nematode (Meloidogyne graminicola) to rice (Oryza sativa L.)

Effectors play key roles in the parasitism of the plant-parasitic nematode. Silencing the effector-coding genes was applied to study the function and role of nematode effectors. In this study, the Mgra16281 gene (ID: MK322955.1) encoding an effector with the unknown function was cloned from the rice root-knot nematode Meloidogyne graminicola isolated in Long An province. To knock-down the expression of this gene, an artificial microRNA was synthesized based on the Osa-MIR528 precursor and inserted into an expression vector. This microRNA can be expressed in rice to investigate the function of MGRA16281 of root-knot nematode via host-induced gene silencing approach (HIGS).

Factors Affecting the Regeneration, via Organogenesis, and the Selection of Transgenic Calli in the Peach Rootstock Hansen 536 (Prunus persica × Prunus amygdalus) to Express an RNAi Construct against PPV Virus

Prunus spp. is one of the most recalcitrant fruit tree species in terms of in vitro regeneration and transformation, mostly when mature tissues are used as explants. The present study describes the in vitro regeneration via indirect organogenesis, and Agrobacterium tumefaciens-mediated transformation of the peach rootstock Hansen 536 (Prunus persica × Prunus amygdalus) through the use of meristematic bulks (MBs) as starting explants. Efficient adventitious shoot regeneration was obtained when Hansen 536 MBs were cultured on an optimized medium consisting of modified McCown Woody Plant medium (WPM) enriched with 4.4 M 6-Benzyladenine (BA), 0.1 M 1-Naphthaleneacetic acid (NAA) and 6.0 g L−1 plant agar S1000 (B&V). MB slices were used later as starting explants for Agrobacterium-mediated transformation to introduce an RNAi construct “ihp35S-PPV194” against PPV virus. Transgenic events were identified by both green fluorescent protein (GFP) screening and kanamycin selection at different concentrations (0, 17 or 42 M). GFP-fluorescent proliferating callus lines were selected and confirmed to stably express the ihp35S-PPV194::eGFP gene construct by molecular analysis. Although shoot regeneration from these transgenic calli has not been obtained yet, this represents one of the few examples of successful attempts in peach genetic transformation from somatic tissues, and also serves as a useful in vitro system for future gene functional analysis in peach.

Silencing cathepsin L expression reduces Myzus persicae protein content and the nutritional value as prey for Coccinella septempunctata

Gut-expressed aphid genes, which may be more easily inhibited by RNA interference (RNAi) constructs, are attractive targets for pest control efforts involving transgenic plants. Here we show that expression of cathepsin L, a cysteine protease that functions in aphid guts, can be reduced by expression of an RNAi construct in transgenic tobacco. The effectiveness of this approach is demonstrated by up to 80% adult mortality, reduced fecundity, and delayed nymph production of Myzus persicae (green peach aphids) when cathepsin L expression was reduced by plant-mediated RNAi. Consistent with the function of cathepsin L as a gut protease, M. persicae fed on the RNAi plants had a lower protein content in their bodies and excreted more protein in their honeydew. Larvae of Coccinella septempunctata (seven-spotted ladybugs) grew more slowly on aphids having reduced cathepsin L expression, suggesting that prey insect nutritive value, and not just direct negative effects of the RNAi construct, needs to be considered when producing transgenic plants for RNAi-mediated pest control.HighlightsSilencing expression of cathepsin L by RNA interference reduces protein content of Myzus persicae (green peach aphid) bodies.Honeydew of aphids with cathepsin L silenced contains elevated protein.Cathepsin L is required for efficient protein uptake from phloem sap.Aphids with cathepsin L expression silenced have increased mortality and fewer offspring.Coccinella septempunctata (seven-spotted ladybugs) grow more slowly on aphids with expression of cathepsin L silenced.