Organization of the TC and TE cellular T‐DNA regions in Nicotiana otophora and functional analysis of three diverged TE‐6b genes

Nicotiana otophora contains Agrobacterium‐derived T‐DNA sequences introduced by horizontal gene transfer (Chen et al., 2014). Sixty‐nine contigs were assembled into four different cellular T‐DNAs (cT‐DNAs) totalling 83 kb. TC and TE result from two successive transformation events, each followed by duplication, yielding two TC and two TE inserts. TC is also found in other Nicotiana species, whereas TE is unique to N. otophora. Both cT‐DNA regions are partially duplicated inverted repeats. Analysis of the cT‐DNA divergence patterns allowed reconstruction of the evolution of the TC and TE regions. TC and TE carry 10 intact open reading frames. Three of these are TE‐6b genes, derived from a single 6b gene carried by the Agrobacterium strain which inserted TE in the N. otophora ancestor. 6b genes have so far only been found in Agrobacterium tumefaciens or Agrobacterium vitis T‐DNAs and strongly modify plant growth (Chen and Otten, 2016). The TE‐6b genes were expressed in Nicotiana tabacum under the constitutive 2 × 35S promoter. TE‐1‐6b‐R and TE‐2‐6b led to shorter plants, dark‐green leaves, a strong increase in leaf vein development and modified petiole wings. TE‐1‐6b‐L expression led to a similar phenotype, but in addition leaves show outgrowths at the margins, flowers were modified and plants became viviparous, i.e. embryos germinated in the capsules at an early stage of their development. Embryos could be rescued by culture in vitro. The TE‐6b phenotypes are very different from the earlier described 6b phenotypes and could provide new insight into the mode of action of the 6b genes.     

A novel receptor‐like kinase involved in fungal pathogen defence in Arabidopsis thaliana

Plants are under constant attack from a variety of disease‐causing organisms. Lacking an adaptive immune system, plants repel pathogen attack via an array of pathogen recognition machinery. Receptor‐like kinases (RLKs) are involved in the recognition of pathogen‐associated molecular patterns (PAMPs) and activate resistance pathways against broad classes of pathogens. We have identified powdery mildew‐resistant kinase 1, an Arabidopsis gene encoding an RLK that is highly induced by chitin at early time points and localizes to the plasma membrane. Knockout mutants in pmrk1 are more susceptible to both Golovinomyces cichoracearum and Plectosphaerella cucumerina. Our data show that PMRK1 is essential in early stages of defence against fungi and provide evidence that PMRK1 may be unique to chitin‐induced signalling pathways. The results of this study indicate that PMRK1 is a critical component of plant innate immunity against fungal pathogens.     

A pair of receptor‐like kinases is responsible for natural variation in shoot growth response to mannitol treatment in Arabidopsis thaliana

Growth is a complex trait that adapts to the prevailing conditions by integrating many internal and external signals. Understanding the molecular origin of this variation remains a challenging issue. In this study, natural variation of shoot growth under mannitol‐induced stress was analyzed by standard quantitative trait locus mapping methods in a recombinant inbred line population derived from a cross between the Col‐0 and Cvi‐0 Arabidopsis thaliana accessions. Cloning of a major QTL specific to mannitol‐induced stress condition led to identification of EGM1 and EGM2, a pair of tandem‐duplicated genes encoding receptor‐like kinases that are potentially involved in signaling of mannitol‐associated stress responses. Using various genetic approaches, we identified two non‐synonymous mutations in the EGM2[Cvi] allele that are shared by at least ten accessions from various origins and are probably responsible for a specific tolerance to mannitol. We have shown that the enhanced shoot growth phenotype contributed by the Cvi allele is not linked to generic osmotic properties but instead to a specific chemical property of mannitol itself. This result raises the question of the function of such a gene in A. thaliana, a species that does not synthesize mannitol. Our findings suggest that the receptor‐like kinases encoded by EGM genes may be activated by mannitol produced by pathogens such as fungi, and may contribute to plant defense responses whenever mannitol is present.     

Characterization of stress‐responsive lncRNAs in Arabidopsis thaliana by integrating expression,epigenetic and structural features

Agrobacterium genetically transforms plants by transferring and integrating T‐(transferred) DNA into the host genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1 (VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction with and targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains. In no instance could we detect altered transformation susceptibility. We also used confocal microscopy to examine the subcellular localization of Venus‐tagged VirE2 or Venus‐tagged VIP1, in the presence or absence of the other untagged protein, in different plant cell systems. We found that VIP1–Venus localized in both the cytoplasm and the nucleus of Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts and tobacco BY‐2 protoplasts, regardless of whether VirE2 was co‐expressed. VirE2 localized exclusively to the cytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpression. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionally detect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. The vast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacterium‐mediated transformation or VirE2 subcellular localization.     

Root‐specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium‐derived T‐DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2′ gene (TB‐mas2′). This gene is similar to the Agrobacterium rhizogenes A4‐mas2′ gene that encodes the synthesis of the Amadori compound deoxyfructosyl‐glutamine (DFG or santhopine). In this study we show that TB‐mas2′ is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB‐mas2′ expression levels. The TB‐mas2′ promoter sequences of low‐ and high‐expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low‐ and a high‐expression cultivar. pTB‐mas2′‐GUS and pA4‐mas2′‐GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB‐mas2′ expression contain detectable levels of DFG.     

Gibberellic acid signaling is required for ambient temperature‐mediated induction of flowering in Arabidopsis thaliana

Distinct molecular mechanisms integrate changes in ambient temperature into the genetic pathways that govern flowering time in Arabidopsis thaliana. Temperature‐dependent eviction of the histone variant H2A.Z from nucleosomes has been suggested to facilitate the expression of FT by PIF4 at elevated ambient temperatures. Here we show that, in addition to PIF4, PIF3 and PIF5, but not PIF1 and PIF6, can promote flowering when expressed specifically in phloem companion cells (PCC), where they can induce FT and its close paralog, TSF. However, despite their strong potential to promote flowering, genetic analyses suggest that the PIF genes seem to have only a minor role in adjusting flowering in response to photoperiod or high ambient temperature. In addition, loss of PIF function only partially suppressed the early flowering phenotype and FT expression of the arp6 mutant, which is defective in H2A.Z deposition. In contrast, the chemical inhibition of gibberellic acid (GA) biosynthesis resulted in a strong attenuation of early flowering and FT expression in arp6. Furthermore, GA was able to induce flowering at low temperature (15°C) independently of FT, TSF, and the PIF genes, probably directly at the shoot apical meristem. Together, our results suggest that the timing of the floral transition in response to ambient temperature is more complex than previously thought and that GA signaling might play a crucial role in this process.     

Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T‐DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana

Nicotiana species carry cellular T‐DNA sequences (cT‐DNAs), acquired by Agrobacterium‐mediated transformation. We characterized the cT‐DNA sequences of the ancestral Nicotiana tabacum species Nicotiana tomentosiformis by deep sequencing. N. tomentosiformis contains four cT‐DNA inserts derived from different Agrobacterium strains. Each has an incomplete inverted‐repeat structure. TA is similar to part of the Agrobacterium rhizogenes 1724 mikimopine‐type T‐DNA, but has unusual orf14 and mis genes. TB carries a 1724 mikimopine‐type orf14‐mis fragment and a mannopine‐agropine synthesis region (mas2‐mas1‐ags). The mas2′ gene codes for an active enzyme. TC is similar to the left part of the A. rhizogenes A4 T‐DNA, but also carries octopine synthase‐like (ocl) and c‐like genes normally found in A. tumefaciens. TD shows a complex rearrangement of T‐DNA fragments similar to the right end of the A4 TL‐DNA, and including an orf14‐like gene and a gene with unknown function, orf511. The TA, TB, TC and TD insertion sites were identified by alignment with N. tabacum and Nicotiana sylvestris sequences. The divergence values for the TA, TB, TC and TD repeats provide an estimate for their relative introduction times. A large deletion has occurred in the central part of the N. tabacum cv. Basma/Xanthi TA region, and another deletion removed the complete TC region in N. tabacum. Nicotiana otophora lacks TA, TB and TD, but contains TC and another cT‐DNA, TE. This analysis, together with that of Nicotiana glauca and other Nicotiana species, indicates multiple sequential insertions of cT‐DNAs during the evolution of the genus Nicotiana.     

Efficacy of a plant‐produced virus‐like particle vaccine in chickens challenged with Influenza A H6N2 virus

The efficacy, safety, speed, scalability and cost‐effectiveness of producing hemagglutinin‐based virus‐like particle (VLP) vaccines in plants are well‐established for human influenza, but untested for the massive poultry influenza vaccine market that remains dominated by traditional egg‐grown oil‐emulsion whole inactivated virus vaccines. For optimal efficacy, a vaccine should be closely antigenically matched to the field strain, requiring that influenza A vaccines be updated regularly. In this study, an H6 subtype VLP transiently expressed in Nicotiana benthamiana was formulated into a vaccine and evaluated for efficacy in chickens against challenge with a heterologous H6N2 virus. A single dose of the plant‐produced H6 VLP vaccine elicited an immune response comparable to two doses of a commercial inactivated H6N2 vaccine, with mean hemagglutination inhibition titres of 9.3 log₂ and 8.8 log₂, respectively. Compared to the non‐vaccinated control, the H6 VLP vaccine significantly reduced the proportion of shedders and the magnitude of viral shedding by >100‐fold in the oropharynx and >6‐fold in the cloaca, and shortened oropharyngeal viral shedding by at least a week. Despite its potency, the cost of the antigenic mismatch between the inactivated H6N2 vaccine and challenge strain was evident not only in this vaccine's failure to reduce viral shedding compared to the non‐vaccinated group, but its apparent exacerbation of oropharyngeal viral shedding until 21 days post‐challenge. We estimate that a kilogram of plant leaf material can produce H6 VLP vaccines sufficient for between 5000 and 30 000 chickens, depending on the effective dose and whether one or two immunizations are administered.     

Differential contributions of plant Dicer‐like proteins to antiviral defences against potato virus X in leaves and roots

Members of the plant Dicer‐like (DCL) protein family are the critical components of the RNA‐silencing pathway that mediates innate antiviral defence. The distinct antiviral role of each individual DCL protein has been established with mostly based on observations of aerial parts of plants. Thus, although the roots are closely associated with the life cycle of many plant viruses, little is known about the antiviral activities of DCL proteins in roots. We observed that antiviral silencing strongly inhibits potato virus X (PVX) replication in roots of some susceptible Solanaceae species. Silencing of the DCL4 homolog in Nicotiana benthamiana partially elevated PVX replication levels in roots. In Arabidopsis thaliana, which was originally considered a non‐host plant of PVX, high levels of PVX accumulation in inoculated leaves were achieved by inactivation of DCL4, while in the upper leaves and roots, it required the additional inactivation of DCL2. In transgenic A. thaliana carrying the PVX amplicon with a green fluorescent protein (GFP) gene insertion in the chromosome (AMP243 line), absence of DCL4 enabled high levels of PVX‐GFP accumulation in various aerial organs but not in the roots, suggesting that DCL4 is critical for intracellular antiviral silencing in shoots but not in roots, where it can be functionally compensated by other DCL proteins. Together, the high level of functional redundancies among DCL proteins may contribute to the potent antiviral activities against PVX replication in roots.     

Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana

Root hairs are specialized cells that are important for nutrient uptake. It is well established that nutrients such as phosphate have a great influence on root hair development in many plant species. Here we investigated the role of nitrate on root hair development at a physiological and molecular level. We showed that nitrate increases root hair density in Arabidopsis thaliana. We found that two different root hair defective mutants have significantly less nitrate than wild‐type plants, suggesting that in A. thaliana root hairs have an important role in the capacity to acquire nitrate. Nitrate reductase‐null mutants exhibited nitrate‐dependent root hair phenotypes comparable with wild‐type plants, indicating that nitrate is the signal that leads to increased formation of root hairs. We examined the role of two key regulators of root hair cell fate, CPC and WER, in response to nitrate treatments. Phenotypic analyses of these mutants showed that CPC is essential for nitrate‐induced responses of root hair development. Moreover, we showed that NRT1.1 and TGA1/TGA4 are required for pathways that induce root hair development by suppression of longitudinal elongation of trichoblast cells in response to nitrate treatments. Our results prompted a model where nitrate signaling via TGA1/TGA4 directly regulates the CPC root hair cell fate specification gene to increase formation of root hairs in A. thaliana.