A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

Mitogen‐activated protein kinase phosphatase 1 reduces the replication efficiency of Bamboo mosaic virus in Nicotiana benthamiana

In plants, the mitogen‐activated protein kinase (MAPK) cascades are the central signaling pathways of the complicated defense network triggered by the perception of pathogen‐associated molecular patterns to repel pathogens. The Arabidopsis thaliana MAPK phosphatase 1 (AtMKP1) negatively regulates the activation of MAPKs. Recently, the AtMKP1 homolog of Nicotiana benthamiana (NbMKP1) was found in association with the Bamboo mosaic virus (BaMV) replication complex. This study aimed to investigate the role of NbMKP1 in BaMV multiplication in N. benthamiana. Silencing of NbMKP1 increased accumulations of the BaMV‐encoded proteins and the viral genomic RNA, although the same condition reduced the infectivity of Pseudomonas syringae pv. tomato DC3000 in N. benthamiana. On the other hand, overexpression of NbMKP1 decreased the BaMV coat protein accumulation in a phosphatase activity‐dependent manner in protoplasts. NbMKP1 also negatively affected the in vitro RNA polymerase activity of the BaMV replication complex. Collectively, the activity of NbMKP1 seems to reduce BaMV multiplication, inconsistent with the negatively regulatory role of MKP1 in MAPK cascades in terms of warding off fungal and bacterial invasion. In addition, silencing of NbMKP1 increased the accumulation of Foxtail mosaic virus but decreased Potato virus X. The discrepant effects exerted by NbMKP1 on different pathogens foresee the difficulty to develop plants with broad‐spectrum resistance through genetically manipulating a single player in MAPK cascades.     

Utility of the P19 suppressor of gene‐silencing protein for production of therapeutic antibodies in Nicotiana expression hosts

To study how the P19 suppressor of gene‐silencing protein can be used effectively for the production of therapeutic glycoproteins, the following factors were examined: the genetic elements used for expressing recombinant proteins; the effect of different P19 concentrations; compatibility of P19 with various Nicotiana tabacum cultivars for transgenic expression; the glycan profile of a recombinant therapeutic glycoprotein co‐expressed with P19 in an RNAi‐based glycomodified Nicotiana benthamiana expression host. The coding sequences for the heavy and light chains of trastuzumab were cloned into five plant expression vectors (102–106) containing different 5′ and 3′ UTRs, designated as vector sets 102–106 mAb. The P19 protein of Tomato bushy stunt virus (TBSV) was also cloned into vector 103, which contained the Cauliflower mosaic virus (CaMV) 35S promoter and 5′UTR together with the terminator region of the nopaline synthase gene of Agrobacterium. Transient expression of the antibody vectors resulted in different levels of trastuzumab accumulation, the highest being 105 and 106 mAb at about 1% of TSP. P19 increased the concentration of trastuzumab approximately 15‐fold (to about 2.3% of TSP) when co‐expressed with 103 mAb but did not affect antibody levels with vectors 102 and 106 mAb. When 103 mAb was expressed together with P19 in different N. tabacum cultivars, all except Little Crittenden showed a marked discolouring of the infiltrated areas of the leaf and decreased antibody expression. Co‐expression of P19 also abolished antibody accumulation in crosses between N. tabacum cv. I‐64 and Little Crittenden, indicating a dominant mode of inheritance for the observed P19‐induced responses.     

Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC‐NBS‐LRR resistance gene of Malus × robusta 5

The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5‐virulent and Mr5‐avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5‐avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5‐virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene‐for‐gene interaction in the host–pathogen relationship Mr5–E. amylovora.     

Target and non‐target effects of a spider venom toxin produced in transgenic cotton and tobacco plants

The peptide ω‐Hexatoxin‐Hv1a (Hvt) is one of the most studied spider toxins. Its insecticidal potential has been reported against species belonging to the arthropod orders Lepidoptera, Diptera and Orthoptera. The gene encoding Hvt has been transformed into cotton and tobacco to protect the plants from damage by lepidopteran pests. This study evaluated the expression of the ω‐HXTX‐Hv1a gene in transgenic plants, and the toxicity of plant‐expressed and purified Hvt on target lepidopteran insects and on several non‐target species. Transgenic Bollgard II cotton plants, which produce Cry1Ac and Cry2Ab2 and purified Cry2Ab2 protein were included in the study as comparators. LC₉₅ values of purified Hvt against Spodoptera littoralis and Heliothis virescens were 28.31 and 27.57 μg/ml of artificial diet, respectively. Larval mortality was 100% on Hvt‐transgenic tobacco plants but not on Hvt‐transgenic cotton, probably because of the significantly lower toxin expression level in the transgenic cotton line. Non‐target studies were conducted with larvae of the predators Chrysoperla carnea and Coccinella septempunctata, adults of the aphid parasitoid Aphidius colemani, and adult workers of the honey bee, Apis mellifera. Even at 40 μg/ml, Hvt did not adversely affect the four non‐target species. Purified Cry2Ab2 at 10 μg/ml also did not adversely affect any of the non‐target species. Our results show that Hvt might be useful for developing insecticidal plant varieties to control pest Lepidoptera.     

The Lr34 adult plant rust resistance gene provides seedling resistance in durum wheat without senescence

The hexaploid wheat (Triticum aestivum) adult plant resistance gene, Lr34/Yr18/Sr57/Pm38/Ltn1, provides broad‐spectrum resistance to wheat leaf rust (Lr34), stripe rust (Yr18), stem rust (Sr57) and powdery mildew (Pm38) pathogens, and has remained effective in wheat crops for many decades. The partial resistance provided by this gene is only apparent in adult plants and not effective in field‐grown seedlings. Lr34 also causes leaf tip necrosis (Ltn1) in mature adult plant leaves when grown under field conditions. This D genome‐encoded bread wheat gene was transferred to tetraploid durum wheat (T. turgidum) cultivar Stewart by transformation. Transgenic durum lines were produced with elevated gene expression levels when compared with the endogenous hexaploid gene. Unlike nontransgenic hexaploid and durum control lines, these transgenic plants showed robust seedling resistance to pathogens causing wheat leaf rust, stripe rust and powdery mildew disease. The effectiveness of seedling resistance against each pathogen correlated with the level of transgene expression. No evidence of accelerated leaf necrosis or up‐regulation of senescence gene markers was apparent in these seedlings, suggesting senescence is not required for Lr34 resistance, although leaf tip necrosis occurred in mature plant flag leaves. Several abiotic stress‐response genes were up‐regulated in these seedlings in the absence of rust infection as previously observed in adult plant flag leaves of hexaploid wheat. Increasing day length significantly increased Lr34 seedling resistance. These data demonstrate that expression of a highly durable, broad‐spectrum adult plant resistance gene can be modified to provide seedling resistance in durum wheat.     

Cowpea mosaic virus chimeric particles bearing the ectodomain of matrix protein 2 (M2E) of the influenza a virus: Production and characterization

The epitope presentation system for the ectodomain of the M2 protein (M2e) of the influenza A virus was constructed on the basis of the cowpea mosaic virus (CPMV) for expression in the plant Vigna unguiculata. CPMV is widely used as a vector to produce immunogenic chimeric virus particles (CVPs) bearing epitopes of various infectious human and animal pathogens. To produce chimeric CPMV particles in plants, two binary vectors were constructed to bear a modified gene coding for the CPMV S-coat protein with insertions of M2e epitopes of human influenza and bird influenza viruses. Antigenic and immunogenic properties of CVPs were investigated in mice immunization experiments. CVPs were shown to induce anti-M2e IgG production and to partly protect mice against a challenge with low doses of the influenza virus. However, low infectivity and immunogenicity of chimeric CPMV particles indicate that the plant virus-based systems for M2e epitope presentation requires further optimization in order to use plants as a possible source of flu vaccines.     

A VIGS screen identifies immunity in the Arabidopsis Pla‐1 accession to viruses in two different genera of the Geminiviridae

Geminiviruses are DNA viruses that cause severe crop losses in different parts of the world, and there is a need for genetic sources of resistance to help combat them. Arabidopsis has been used as a source for virus‐resistant genes that derive from alterations in essential host factors. We used a virus‐induced gene silencing (VIGS) vector derived from the geminivirus Cabbage leaf curl virus (CaLCuV) to assess natural variation in virus–host interactions in 190 Arabidopsis accessions. Silencing of CH‐42, encoding a protein needed to make chlorophyll, was used as a visible marker to discriminate asymptomatic accessions from those showing resistance. There was a wide range in symptom severity and extent of silencing in different accessions, but two correlations could be made. Lines with severe symptoms uniformly lacked extensive VIGS, and lines that showed attenuated symptoms over time (recovery) showed a concomitant increase in the extent of VIGS. One accession, Pla‐1, lacked both symptoms and silencing, and was immune to wild‐type infectious clones corresponding to CaLCuV or Beet curly top virus (BCTV), which are classified in different genera in the Geminiviridae. It also showed resistance to the agronomically important Tomato yellow leaf curl virus (TYLCV). Quantitative trait locus mapping of a Pla‐1 X Col‐0 F₂ population was used to detect a major peak on chromosome 1, which is designated gip‐1 (geminivirus immunity Pla‐1‐1). The recessive nature of resistance to CaLCuV and the lack of obvious candidate genes near the gip‐1 locus suggest that a novel resistance gene(s) confers immunity.     

Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.     

A thioredoxin NbTRXh2 from Nicotiana benthamiana negatively regulates the movement of Bamboo mosaic virus

An up‐regulated gene derived from Bamboo mosaic virus (BaMV)‐infected Nicotiana benthamiana plants was cloned and characterized in this study. BaMV is a single‐stranded, positive‐sense RNA virus. This gene product, designated as NbTRXh2, was matched with sequences of thioredoxin h proteins, a group of small proteins with a conserved active‐site motif WCXPC conferring disulfide reductase activity. To examine how NbTRXh2 is involved in the infection cycle of BaMV, we used the virus‐induced gene silencing technique to knock down NbTRXh2 expression in N. benthamiana and inoculated the plants with BaMV. We observed that, compared with control plants, BaMV coat protein accumulation increased in knockdown plants at 5 days post‐inoculation (dpi). Furthermore, BaMV coat protein accumulation did not differ significantly between NbTRXh2‐knockdown and control protoplasts at 24 hpi. The BaMV infection foci in NbTRXh2‐knockdown plants were larger than those in control plants. In addition, BaMV coat protein accumulation decreased when NbTRXh2 was transiently expressed in plants. These results suggest that NbTRXh2 plays a role in restricting BaMV accumulation. Moreover, confocal microscopy results showed that NbTRXh2‐OFP (NbTRXh2 fused with orange fluorescent protein) localized at the plasma membrane, similar to AtTRXh9, a homologue in Arabidopsis. The expression of the mutant that did not target the substrates failed to reduce BaMV accumulation. Co‐immunoprecipitation experiments revealed that the viral movement protein TGBp2 could be the target of NbTRXh2. Overall, the functional role of NbTRXh2 in reducing the disulfide bonds of targeting factors, encoded either by the host or virus (TGBp2), is crucial in restricting BaMV movement.     

A phosphopantetheinyl transferase that is essential for mitochondrial fatty acid biosynthesis

In this study we report the molecular genetic characterization of the Arabidopsis mitochondrial phosphopantetheinyl transferase (mtPPT), which catalyzes the phosphopantetheinylation and thus activation of mitochondrial acyl carrier protein (mtACP) of mitochondrial fatty acid synthase (mtFAS). This catalytic capability of the purified mtPPT protein (encoded by AT3G11470) was directly demonstrated in an in vitro assay that phosphopantetheinylated mature Arabidopsis apo‐mtACP isoforms. The mitochondrial localization of the AT3G11470‐encoded proteins was validated by the ability of their N‐terminal 80‐residue leader sequence to guide a chimeric GFP protein to this organelle. A T‐DNA‐tagged null mutant mtppt‐1 allele shows an embryo‐lethal phenotype, illustrating a crucial role of mtPPT for embryogenesis. Arabidopsis RNAi transgenic lines with reduced mtPPT expression display typical phenotypes associated with a deficiency in the mtFAS system, namely miniaturized plant morphology, slow growth, reduced lipoylation of mitochondrial proteins, and the hyperaccumulation of photorespiratory intermediates, glycine and glycolate. These morphological and metabolic alterations are reversed when these plants are grown in a non‐photorespiratory condition (i.e. 1% CO₂ atmosphere), demonstrating that they are a consequence of a deficiency in photorespiration due to the reduced lipoylation of the photorespiratory glycine decarboxylase.     

Impacts of nitrogen fertilizer on major insect pests and their predators in transgenic Bt rice lines T2A‐1 and T1C‐19

Nitrogen is a critical factor for plant development and nitrogen input is one of the important tactics to enhance the development and yield of crops. Nevertheless, nitrogen input could influence the occurrence of insects positively or negatively. Nitrogen is also one of the main elements composing the insecticidal crystal (Cry) protein. Cry protein production could affect nitrogen partitioning in Bt plants and as such nitrogen input may influence insect pest management in transgenic Bt rice, Oryza sativa L. (Poaceae). To test this possibility, we evaluated the impacts of nitrogen regimes on the main insect pests and their predators on two Bt rice lines, T2A‐1 and T1C‐19, expressing Cry2A and Cry1C, respectively, and their non‐transgenic parental counterpart MH63. The results showed that Cry proteins with different nitrogen regimes have enough insecticidal activity on rice leaffolder, Cnaphalocrocis medinalis Guenée (Lepidoptera: Crambidae), in both laboratory and field experiments. Laboratory studies indicated that relevant parameters of ecological fitness in brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), a non‐target insect pest, were significantly affected by nitrogen input both on Bt and MH63 rice lines. Nymphal survival, female adult longevity, and egg hatchability in N. lugens differed significantly among rice varieties. The experiments conducted in rice fields also demonstrated that nitrogen was positively correlated with the abundance of N. lugens on Bt rice, similar to that on MH63 rice. The abundances of two predators – the wolf spider Pirata subpiraticus (Boesenberg & Strand) (Araneae: Lycosidae) and the bug Cyrthorhinus lividipennis Reuter (Hemiptera: Miridae) – were significantly affected by rice growth stages but not by nitrogen input and rice varieties. In conclusion, the above results indicate that high nitrogen regimes for Bt rice (T2A‐1 and T1C‐19) and non‐Bt rice (MH63) cannot facilitate the management of insect pests.     

Spike‐dip transformation of Setaria viridis

Traditional method of Agrobacterium‐mediated transformation through the generation of tissue culture had limited success for Setaria viridis, an emerging C4 monocot model. Here we present an efficient in planta method for Agrobacterium‐mediated genetic transformation of S. viridis using spike dip. Pre‐anthesis developing spikes were dipped into a solution of Agrobacterium tumefaciens strain AGL1 harboring the β‐glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S (CaMV35S) promoter to standardize and optimize conditions for transient as well as stable transformations. A transformation efficiency of 0.8 ± 0.1% was obtained after dipping of 5‐day‐old S3 spikes for 20 min in Agrobacterium cultures containing S. viridis spike‐dip medium supplemented with 0.025% Silwet L‐77 and 200 μm acetosyringone. Reproducibility of this method was demonstrated by generating stable transgenic lines expressing β‐glucuronidase plus (GUSplus), green fluorescent protein (GFP) and Discosoma sp. red fluorescent protein (DsRed) reporter genes driven by either CaMV35S or intron‐interrupted maize ubiquitin (Ubi) promoters from three S. viridis genotypes. Expression of these reporter genes in transient assays as well as in T1 stable transformed plants was monitored using histochemical, fluorometric GUS activity and fluorescence microscopy. Molecular analysis of transgenic lines revealed stable integration of transgenes into the genome, and inherited transgenes expressed in the subsequent generations. This approach provides opportunities for the high‐throughput transformation and potentially facilitates translational research in a monocot model plant.     

Overexpression of AtPAD4 in transgenic Brachypodium distachyon enhances resistance to Puccinia brachypodii

• Brachypodium distachyon (L.) has recently emerged as a model for temperate grasses for investigating the molecular basis of plant–pathogen interactions. Phytoalexin deficient 4 (PAD4) plays a regulatory role in mediating expression of genes involved in plant defence.
• In this research, we generated transgenic B. distachyon plants constitutively overexpressing AtPAD4. Two transgenic B. distachyon lines were verified using PCR and GUS phenotype.
• Constitutive expression of AtPAD4 in B. distachyon enhanced resistance to Puccinia brachypodii. P. brachypodii generated less urediniospores on transgenic than on wild‐type plants. AtPAD4 overexpression enhanced salicylic acid (SA) levels in B. distachyon‐infected tissues. qRT‐PCR showed that expression of pathogenesis‐related 1 (PR1) and other defence‐related genes were up‐regulated in transformed B. distachyon following infection with P. brachypodii.
• Our results indicate that AtPAD4 overexpression in B. distachyon plants led to SA accumulation and induced PR gene expression that reduced the rate of colonisation by P. brachypodii.

     

Jasmonate‐dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell‐wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell‐wall composition to reinforce this defensive barrier remains unknown. The enzyme 13–allene oxide synthase (13–AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13–AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13–AOS enzymes. Indeed, transgenic potato plants lacking both St13–AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound‐responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild‐type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell‐wall pectin composition between wild‐type and CoAOS1/2 plants. Importantly, wild‐type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii.