Interaction of VvDELLA2 and VvCEB1 Mediates Expression of Expansion-Related Gene during GA-Induced Enlargement of Grape Fruit.

Exogenous gibberellin treatment can promote early growth of grape fruit, but the underlying regulatory mechanisms are not well understood. Here, we show that VvDELLA2 directly regulates the activity of the VvCEB1 transcription factor, a key regulator in the control of cell expansion in grape fruit. Our results show that VvCEB1 binds directly to the promoters of cell expansion-related genes in grape fruit and acts as a transcriptional activator, while VvDELLA2 blocks VvCEB1 function by binding to its activating structural domain. The exogenous gibberellin treatment relieved this inhibition by promoting the degradation of VvDELLA2 protein, thus, allowing VvCEB1 to transcriptionally activate the expression of cell expansion-related genes. In conclusion, we conclude that exogenous GA3 treatment regulates early fruit expansion by affecting the VvDELLA-VvCEB1 interaction in grape fruit development.

The fungal elicitor eutypine from Eutypa lata activates basal immunity through its phenolic side chains.

Grapevine trunk diseases (GTDs) affect grape production and reduce vineyard longevity worldwide. Since the causative fungi also occur in asymptomatic trunks, we address disease outbreak in terms of altered chemical communication between host and endophyte. Here, we identified four chemically similar secondary metabolites secreted by the GTD-associated fungus Eutypa lata to analyse their modes of action in a grapevine cell culture of Vitis rupestris, where microtubules were tagged by GFP. Treatment with the metabolite eutypine activated defence responses, evident from extracellular alkalinisation and induction of defence genes. Eutypinol, instead, eliminated microtubules, in contrast to the other three compounds. Furthermore, we evaluated the effect of four corresponding chemical analogues of these compounds, sharing the phenolic but lacking the alkyne moiety. These analogues were able to induce similar defence responses in V. rupestris cells, albeit at reduced amplitude. Since closely related moieties differing only in details of the side groups at the phenolic ring differ significantly with respect to the response of the host cell, we propose that these fungal compounds act through a specific binding site at the membrane of grapevine cells. We corroborate this specificity by combination experiments, where the eutypine and the eutypinol analogues behave competitively with respect to the elicited responses. In summary, Eutypa lata secretes compounds that elicit host defence in a specific manner by interfering with early events of immunity signalling. This supports the notion that a real understanding of GTDs has to address inter-organismic chemical communication.

Dissecting the membrane-microtubule sensor in grapevine defence.

Specific populations of plant microtubules cooperate with the plasma membrane to sense and process abiotic stress signals, such as cold stress. The current study derived from the question, to what extent this perception system is active in biotic stress signalling. The experimental system consisted of grapevine cell lines, where microtubules or actin filaments are visualised by GFP, such that their response became visible in vivo. We used the bacterial elicitors harpin (inducing cell-death related defence), or flg22 (inducing basal immunity) in combination with modulators of membrane fluidity, or microtubules. We show that DMSO, a membrane rigidifier, can cause microtubule bundling and trigger defence responses, including activation of phytoalexin transcripts. However, DMSO inhibited the gene expression in response to harpin, while promoting the gene expression in response to flg22. Treatment with DMSO also rendered microtubules more persistent to harpin. Paradoxically, Benzylalcohol (BA), a membrane fluidiser, acted in the same way as DMSO. Neither GdCl3, nor diphenylene iodonium were able to block the inhibitory effect of membrane rigidification on harpin-induced gene expression. Treatment with taxol stabilised microtubule against harpin but amplified the response of PAL transcripts. Therefore, the data support implications of a model that deploys specific responses to pathogen-derived signals.

Anti-microtubule activity of the traditional Chinese medicine herb Northern Ban Lan (Isatis tinctoria) leads to glucobrassicin.

Traditional Chinese medicine (TCM) belongs to the most elaborate and extensive systems of plant-based healing. The herb Northern Ban Lan (Isatis tinctoria) is famous for its antiviral and anti-inflammatory activity. Although numerous components isolated from I. tinctoria have been characterized so far, their modes of action have remained unclear. Here, we show that extracts from I. tinctoria exert anti-microtubular activity. Using time-lapse microscopy in living tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cells expressing green fluorescent protein-tubulin, we use activity-guided fractionation to screen out the biologically active compounds of I. tinctoria. Among 54 fractions obtained from either leaves or roots of I. tinctoria by methanol (MeOH/H2 O 8:2), or ethyl acetate extraction, one specific methanolic root fraction was selected, because it efficiently and rapidly eliminated microtubules. By combination of further purification with ultra-high-performance liquid chromatography and high-resolution tandem mass spectrometry most of the bioactivity could be assigned to the glucosinolate compound glucobrassicin. Glucobrassicin can also affect microtubules and induce apoptosis in HeLa cells. In the light of these findings, the antiviral activity of Northern Ban Lan is discussed in the context of microtubules being hijacked by many viral pathogens for cell-to-cell spread.

Overexpression of PsoRPM3, an NBS-LRR gene isolated from myrobalan plum, confers resistance to Meloidogyne incognita in tobacco.

KEY MESSAGES: We reported an NBS-LRR gene, PsoRPM3, is highly expressed following RKN infection, initiating an HR response that promotes plant resistance. Meloidogyne spp. are root-knot nematodes (RKNs) that cause substantial economic losses worldwide. Screening for resistant tree resources and identifying plant resistance genes is currently the most effective way to prevent RKN infestations. Here, we cloned a novel TIR-NB-LRR-type resistance gene, PsoRPM3, from Xinjiang wild myrobalan plum (Prunus sogdiana Vassilcz.) and demonstrated that its protein product localized to the nucleus. In response to Meloidogyne incognita infection, PsoRPM3 gene expression levels were significantly higher in resistant myrobalan plum plants compared to susceptible plants. We investigated this difference, discovering that the - 309 to - 19 bp region of the susceptible PsoRPM3 promoter was highly methylated. Indeed, heterologous expression of PsoRPM3 significantly enhanced the resistance of susceptible tobacco plants to M. incognita. Moreover, transient expression of PsoRPM3 induced a hypersensitive response in tobacco, whereas RNAi-mediated silencing of PsoRPM3 in transgenic tobacco reduced this hypersensitive response. Several hypersensitive response marker genes were considerably up-regulated in resistant myrobalan plum plants when compared with susceptible counterparts inoculated with M. incognita. PsoPR1a (a SA marker gene), PsoPR2 (a JA marker gene), and PsoACS6 (an ET signaling marker gene) were all more highly expressed in resistant than in susceptible plants. Together, these results support a model in which PsoRPM3 is highly expressed following RKN infection, initiating an HR response that promotes plant resistance through activated salicylic acid, jasmonic acid, and ethylene signaling pathways.

Transcription Factor Pso9TF Assists Xinjiang Wild Myrobalan Plum (Prunus sogdiana) PsoRPM3 Disease Resistance Protein to Resist Meloidogyne incognita.

The root-knot nematode (Meloidogyne incognita) causes huge economic losses in the agricultural industry throughout the world. Control methods against these polyphagous plant endoparasites are sparse, the preferred one being the deployment of plant cultivars or rootstocks bearing resistance genes against Meloidogyne species. Our previous study has cloned one resistance gene, PsoRPM3, from Xinjiang wild myrobalan plum (Prunus sogdiana). However, the function of PsoRPM3 remains elusive. In the present study, we have investigated the regulatory mechanism of PsoRPM3 in plant defense responses to M. incognita. Our results indicate that fewer giant cells were detected in the roots of the PsoRPM3 transgenic tobacco than wild tobacco lines after incubation with M. incognita. Transient transformations of full-length and TN structural domains of PsoRPM3 have induced significant hypersensitive responses (HR), suggesting that TIR domain might be the one which caused HR. Further, yeast two-hybrid results revealed that the full-length and LRR domain of PsoRPM3 could interact with the transcription factor Pso9TF. The addition of Pso9TF increased the ROS levels and induced HR. Thus, our data revealed that the LRR structural domain of PsoRPM3 may be associated with signal transduction. Moreover, we did not find any relative inductions of defense-related genes PsoEDS1, PsoPAD4 and PsoSAG101 in P. sogdiana, which has been incubated with M. incognita. In summary, our work has shown the key functional domain of PsoRPM3 in the regulation of defense responses to M. incognita in P. sogdiana.

Genome-wide analysis of the Universal stress protein A gene family in Vitis and expression in response to abiotic stress.

Universal Stress Protein A (USPA) plays critical roles in the regulation of growth, development and response to abiotic stress in plants. To date, most research related to the role of USPA in plants has been carried out in herbaceous models such as Arabidopsis, rice and soybean. Here, we used bioinformatics approaches to identify 21 USPA genes in the genome of Vitis vinifera L. Phylogenetic analysis revealed that VvUSPAs could be divided into eight clades. Based on predicted chromosomal locations, we identified 16 pairs of syntenic, orthologous genes between A. thaliana and V. vinifera. Further promoter cis-elements analysis, together with identification of potential microRNA (miRNA) binding sites, suggested that at least some of the VvUSPAs participate in response to phytohormones and abiotic stress. To add support for this, we analyzed the developmental and stress-responsive expression patterns of the homologous USPA genes in the drought-resistant wild Vitis yeshanensis accession 'Yanshan-1' and the drought-sensitive Vitis riparia accession 'He'an'. Most of the USPA genes were upregulated in different degrees in the two genotypes after drought stress and exposure to ethephon (ETH), abscisic acid (ABA) and methyl jasmonate (MeJA). Individual USPA genes showed various tissue-specific expression patterns. Heterologous expression of five selected genes (VvUSPA2, VvUSPA3, VvUSPA11, VvUSPA13 and VvUSPA16) in Escherichia coli (E. coli) enhanced resistance to drought stress. Our study provides a model for mapping gene function in response to abiotic stress and identified three candidate genes, VvUSPA3, VvUSPA11 and VvUSPA16, as regulators of drought response in V. vinifera.

4-CPA (4-Chlorophenoxyacetic Acid) Induces the Formation and Development of Defective "Fenghou" (Vitis vinifera × V. labrusca) Grape Seeds.

For some horticultural plants, auxins can not only induce normal fruit setting but also form fake seeds in the induced fruits. This phenomenon is relatively rare, and, so far, the underlying mechanism remains unclear. In this study, "Fenghou" (Vitis vinifera × V. labrusca) grapes were artificially emasculated before flowering and then sprayed with 4-CPA (4-chlorophenoxyacetic acid) to analyze its effect on seed formation. The results show that 4-CPA can induce normal fruit setting in "Fenghou" grapes. Although more seeds were detected in the fruits of the 4-CPA-treated grapevine, most seeds were immature. There was no significant difference in the seed shape; namely, both fruit seeds of the grapevines with and without 4-CPA treatment contained a hard seed coat. However, the immature seeds lacked embryo and endosperm tissue and could not germinate successfully; these were considered defective seeds. Tissue structure observation of defective seeds revealed that a lot of tissue redifferentiation occurred at the top of the ovule, which increased the number of cell layers of the outer integument; some even differentiated into new ovule primordia. The qRT-PCR results demonstrated that 4-CPA application regulated the expression of the genes VvARF2 and VvAP2, which are associated with integument development in "Fenghou" grape ovules. Together, this study evokes the regulatory role of 4-CPA in the division and continuous redifferentiation of integument cells, which eventually develop into defective seeds with thick seed coats in grapes.

MADS-Box Protein Complex VvAG2, VvSEP3 and VvAGL11 Regulates the Formation of Ovules in Vitis vinifera L. cv. 'Xiangfei'.

The phenomenon of multi-carpel and multi-ovule exists in the grapevine cultivar 'Xiangfei', but the mechanism of ovule formation is seldom reported. In this study, we observed the ovule formation process by using 'Xiangfei' grapes. The role of the VvAG2 (VvAGAMOUS) gene in ovule formation was identified, and we explored the relationship between VvAG2, VvSEP3(VvMADS4) and VvAGL11(VvMADS5) proteins. The results showed that the ovule primordium appeared when the inflorescence length of 'Xiangfei' grapes were 4-5 cm long; the relative expression levels of VvAG2, VvAGL11 and VvSEP3 genes were higher during ovule formation, and the expression levels of VvAG2 gene was the highest. Transgenic tomato (Solanum lycopersicum) plants expressing VvAG2 produced higher numbers of ovules and carpels than the wild type. Moreover, yeast two-hybrid and yeast three-hybrid experiments demonstrated that VvSEP3 acts as a bridge and interacts with VvAG2 and VvAGL11 proteins, respectively. Meanwhile, a homodimer can be formed between VvSEP3 and VvSEP3, but there was no interaction between VvAG2 and VvAGL11. These findings suggest that the VvAG2 gene is involved in the formation of ovules, and VvAG2/VvSEP3 together with VvAGL11/VvSEP3 can form a tetrameric complex. In summary, our data showed that VvAG2 along with VvSEP3 and VvAGL11 jointly regulate the ovule formation of 'Xiangfei' grapes.

Review: Microtubules monitor calcium and reactive oxygen species signatures in signal transduction.

Signal transductions require calcium (Ca2+) or reactive oxygen species (ROS) signatures, which act as chemical and electrical signals in response to various biotic and abiotic stresses. Calcium as an ion or second messenger affects the membrane potential and microtubules (MTs) dynamicity, while MTs can modulate auto-propagating waves of calcium and ROS signatures in collaboration with ion channels depending on the stimulus type. Thus, in the current review, we highlight advances in research focused on the relationship between dynamic MTs and calcium and ROS signatures in short-distance transmission. The challenges of Ca2+-MTs-ROS crosstalk in cold sensing are addressed, which could suggest the prioritization of ROS or Ca2+ in signalling.

The VvSUPERMAN-like Gene Is Differentially Expressed between Bicarpellate and Tricarpellate Florets of Vitis vinifera L. Cv. 'Xiangfei' and Its Heterologous Expression Reduces Carpel Number in Tomato.

Multicarpellate fruits are larger and produce more seeds than mono- or bicarpellate fruits, enhancing the reproductive capacity of the plant. To identify the phenotypic and molecular differences among florets of different carpel types, we studied carpel formation and fusion in the grapevine (Vitis vinifera) cultivar 'Xiangfei', which produces a high proportion of multicarpellate fruit. We also determined the function of VvSUPERMAN-like (VvSUP-like) and explored its relationship with VvWUS (VvWUSCHEL) and VvAG1 (VvAGAMOUS), which is related to the formation of carpel primordia. We showed that carpel formation and fusion were largely consistent between bicarpellate and tricarpellate ovaries, which both involve congenital fusion; rather, the differences between these ovary types arose from variation in carpel primordia number and location. Transgenic tomato (Solanum lycopersicum) plants expressing VvSUP-like produced significantly fewer carpels and other floral organs than the wild type. Moreover, transcriptome sequencing results indicate that VvSUP-like was more highly expressed in bicarpellate than in tricarpellate 'Xiangfei' florets. Luciferase reporter assays indicated that VvSUP-like inhibits the expression of VvAG1 and VvWUS by directly binding to their promoters, and VvWUS promotes VvAG1 expression by directly binding to its promoter. VvSUP-like inhibits the feedback signaling between VvWUS and VvAG1. Together, these results suggest that VvSUP-like negatively regulates the number of carpels that develop by inhibiting VvAG1 and VvWUS expression.

CCS52 and DEL1 function in root-knot nematode giant cell development in Xinjiang wild myrobalan plum (Prunus sogdiana Vassilcz).

Root-knot nematodes (RKNs) are highly invasive plant parasites that establish permanent feeding sites within the roots of the host plant. Successful establishment of the feeding site is essential for the survival of RKN. The formation and development of the feeding cell, also called giant cell, involve both cell division and endoreduplication. Here, we examined giant cell development and endoreduplication in Prunus sogdiana infected with the RKN. We found that feeding sites were established 3-5 days post inoculation (dpi) and matured at 21-28 dpi. The giant cells began to form 5 dpi and continued to increase in size from 7 to 21 dpi. The large numbers of dividing nuclei were observed in giant cells from 7 to 14 dpi. However, nuclear division was rarely observed after 28 days. RT-PCR and in situ hybridization analyses revealed that PsoCCS52A was abundantly expressed at 7-21 dpi and the PsoCCS52A signal observed in giant cell nucleus at 7-14 dpi. The PsoCCS52B is highly expressed at 14 dpi, and the hybridization signal was mainly in the cytoplasm of giant cells. The PsoDEL1 expression was lowest 7-21 dip, with negligible transcript detected in the giant cells. This indicates that the PsoCCS52A plays a role in the process of cell division, while the CCS52B plays a role in the development of giant cells. The PsoDEL1 plays a negative regulatory role in megakaryocyte nuclear replication. These data suggest that an increased expression of PsoCCS52A promotes nuclear division and produces a large number of polyploid nuclei, the area of giant cells and feeding sites increase, ultimately leading to the formation of galls in Prunus sogdiana.

Hunting modulators of plant defence: the grapevine trunk disease fungus Eutypa lata secretes an amplifier for plant basal immunity.

Grapevine trunk diseases (GTDs) are progressively affecting vineyard longevity and productivity worldwide. To be able to understand and combat these diseases, we need a different concept of the signals exchanged between the grapevine and fungi than the well-studied pathogen-associated molecular pattern and effector concepts. We screened extracts from fungi associated with GTDs for their association with basal defence responses in suspension cells of grapevine. By activity-guided fractionation of the two selected extracts, O-methylmellein was identified as a candidate modulator of grapevine immunity. O-Methylmellein could not induce immune responses by itself (i.e. does not act as an elicitor), but could amplify some of the defence responses triggered by the bacterial elicitor flg22, such as the induction level of defence genes and actin remodelling. These findings show that Eutypa lata, exemplarily selected as an endophytic fungus linked with GTDs, can secrete compounds that act as amplifiers of basal immunity. Thus, in addition to elicitors that can trigger basal immunity, and effectors that down-modulate antibacterial basal immunity, once it had been activated, E. lata seems to secrete a third type of chemical signal that amplifies basal immunity and may play a role in the context of consortia of mutually competing microorganisms.