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bioRxiv2017

Heterologous expression of the immune receptor EFR in Medicago truncatula reduces pathogenic infection, but not rhizobial symbiosis

Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad-spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen-associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae-specific PRR ELONGATION FACTOR-THERMO UNSTABLE RECEPTOR (EFR) from Arabidopsis thaliana to the legume Medicago truncatula, conferring recognition of the bacterial EF-Tu protein. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF-Tu-derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR-Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.\n\nSignificance StatementCrop engineering helps reducing the economic and environmental costs of plant disease. The genetic transfer of immune receptors across plant species is a promising biotechnological approach to increase disease resistance. Surface-localized pattern-recognition receptors (PRRs), which detect conserved characteristic microbial features, are functional in heterologous taxonomically-diverse plant species, and confer broad-spectrum disease resistance. It was unclear whether PRR transfer negatively impacts the association of the recipient plants with symbiotic microbes. Here, we show that a legume engineered with a novel PRR recognizing a conserved bacterial protein becomes more resistant to an important bacterial pathogen without significant impact on nitrogen-fixing symbiosis with rhizobia. This finding is of particular relevance as attempts to transfer this important symbiosis into non-legume plants are ongoing.

plant biology↗

bioRxiv2017

A dual sgRNA approach for functional genomics in Arabidopsis thaliana

Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows to generate knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain Cas9 null-segregants with bi-allelic mutations in the T2 generation. Out of seven new mutant alleles we report here, one allele for GRXS17, the ortholog of human GRX3/PICOT, did not phenocopy previously characterized nulls. Notwithstanding, the mutation caused a frameshift and triggered nonsense-mediated decay. We demonstrate that our workflow is also compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method can result in a more reliable loss-of-function alleles that lack a large essential part of the gene. The ease in the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.\n\nOne-sentence summaryWe present a dual sgRNA approach to delete Arabidopsis gene 34 fragments in order to obtain reliable functional knock-outs.

plant biology↗

bioRxiv2017

Molecular machinery of auxin synthesis, secretion, and perception in the unicellular chlorophyte alga Chlorella sorokiniana UTEX 1230

Indole-3-acetic acid is a ubiquitous small molecule found in all domains of life. It is the predominant and most active auxin in seed plants, where it coordinates a variety of complex growth and development processes. The potential origin of auxin signaling in algae remains a matter of some controversy. In order to clarify the evolutionary context of algal auxin signaling, we undertook a genomic survey to assess whether auxin acts as a signaling molecule in the emerging model chlorophyte Chlorella sorokiniana UTEX 1230. C. sorokiniana produces the auxin indole-3-acetic acid (IAA), which was present in both the cell pellet and in the supernatant at a concentration of ~ 1 nM, and its genome encodes orthologs of genes related to auxin synthesis, transport, and signaling in higher plants. Candidate orthologs for the canonical AUX/IAA signaling pathway were not found; however, auxin-binding protein 1 (ABP1), an alternate auxin receptor, is present and highly conserved at essential auxin binding and zinc coordinating residues. Additionally, candidate orthologs for PIN proteins, responsible for intercellular, vectorial auxin transport in higher plants, were not found, but PILs (PIN-Like) proteins, a recently discovered family that mediates intracellular auxin transport, were identified. The distribution of auxin related gene in this unicellular chlorophyte demonstrates that a core suite of auxin signaling components was present early in the evolution of plants. Understanding the simplified auxin signaling pathways in chlorophytes will aid in understanding phytohormone signaling and crosstalk in seed plants, and in understanding the diversification and integration of developmental signals during the evolution of multicellular plants.

plant biology↗

bioRxiv2017

Response of oriental xerophytes to the occidental industrial revolution

Since the Industrial Revolution, human activities have contributed substantially to climate change, by adding CO2 to the atmosphere, especially since the mid-20th century (the \"Great Acceleration\"). Climatic change does not have the same impact on different regions of the Earth, neither in the recorded past, nor in models of the future. Therefore, to anticipate on these changes, we need to understand and be able to predict the possible responses of the different regional vegetations of the world to these changes, and most significantly to increased drought conditions. The aim of this study is to understand the response of the xerophytes of the immense oriental Irano-Turanian bioregion to post-industrial global warming and to compare it with the response of the xerophytes of the neighbouring occidental Mediterranean bioregion. We measured stomata index and stomata density from 83 herbarium sheets (coll. 1821-2014) from species of the non-succulent xerophyte, Haplophyllum. We tested for differences before and after the \"Great Acceleration\" in both bioregions. SI decreased in the occidental species (significant only for abaxial leaf side), whereas SI significantly increased in the oriental species (both sides). We suggest that changes in both occidental and oriental species are linked to atmospheric CO2 due to the different constraints that act on their growth. In light-limited occidental species, atmospheric CO2 caused the stomata index decrease, whereas in the predominantly water-limited oriental species, increased drought stress and temperature (climatic change) caused stomata index increase. In conclusion, we propose that whereas atmospheric CO2 directly caused a decrease in stomata index in occidental xerophytes, it indirectly caused an increase in stomata index in oriental xerophytes via climate change (increase in aridity, drought stress, and temperature). This study highlights the considerable potential of research based on historical herbarium collections to answer ecological questions, especially regarding climatic change.

plant biology↗

bioRxiv2017

Maternal RNA-directed DNA methylation is required for seed development in Brassica rapa

Some organisms deploy small RNAs from accessory cells to maintain genome integrity in the zygote, a mechanism that has been proposed but not demonstrated in plants. Here we show that maternal mutations in the Pol IV-dependent small RNA pathway cause abortion of developing seeds in Brassica rapa. Surprisingly, small RNA production is required in maternal somatic tissues, but not in maternal gametes or the developing zygote. We propose that parental influence over zygotic genomes is a common strategy in eukaryotes and that outbreeding species such as B. rapa are key to understanding the role of small RNAs during reproduction.

plant biology↗

bioRxiv2017

Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times

- Pennycress (Thlaspi arvense L.) is currently being developed as a new cold-season oilseed crop. Like many Brassicaceae, pennycress can exhibit either a winter or spring annual phenotype. In Arabidopsis, mutations in negative regulators of flowering, including FLOWERING LOCUS C (FLC) and FRIGIDA can cause the transition to a spring annual habit. The genetics underlying the difference between spring and winter annual pennycress are currently unknown.\n\n- Using whole genome sequencing across wild spring annual pennycress accessions, co-segregation analyses, and comparative genomics approaches, we identify new alleles of TaFLC and explore their geographic distribution.\n\n- We report that loss of function mutations in TaFLC confer the spring annual phenotype in pennycress. We have also identified four natural alleles of TaFLC that confer a spring annual growth habit. The two spring annual FLC alleles present in European accessions were only identified in accessions collected in Montana, USA.\n\n- In pennycress, the spring annual habit has arisen several independent times. Accessions harboring the two European alleles were introduced to North America, likely after the species became a widespread on the continent. These findings provide new information on the natural history of the introduction and spread of the spring annual phenotype in pennycress.

plant biology↗

bioRxiv2017

The roles of Conserved Domains in DEMETER-Mediated Active DNA Demethylation in planta

DNA methylation plays critical roles in maintaining genome stability, genomic imprinting, transposon silencing, and development. In Arabidopsis genomic imprinting is established in the central cell by DEMETER (DME)-mediated active DNA demethylation, and is essential for seed viability. DME is a large polypeptide with multiple poorly characterized conserved domains. Here we show that the C-terminal enzymatic core of DME is sufficient to complement dme associated developmental defects. When targeted by a native DME promoter, nuclear-localized DME C-terminal region rescues dme seed abortion and pollen germination defects, and ameliorates CG hypermethylation phenotype in dme-2 endosperm. Furthermore, targeted expression of the DME N-terminal region in wild-type central cell induces dme-like seed abortion phenotype. Our results support a bipartite organization for DME protein, and suggest that the N-terminal region might have regulatory function such as assisting in DNA binding and enhancing the processivity of active DNA demethylation in heterochromatin targets.

plant biology↗

bioRxiv2017

Flavonol rhamnosylation indirectly modifies the cell wall defects of RHAMNOSE BIOSYNTHESIS1 mutants by altering rhamnose flux

Rhamnose is required in Arabidopsis thaliana for synthesizing pectic polysaccharides and glycosylating flavonols. RHAMNOSE BIOSYNTHESIS1 (RHM1) encodes a UDP-L-rhamnose synthase, and rhm1 mutants exhibit many developmental defects, including short root hairs, hyponastic cotyledons, and left-handed helically twisted petals and roots. It has been proposed that the hyponastic cotyledons observed in rhm1 mutants are a consequence of abnormal flavonol glycosylation, while the root hair defect is flavonol-independent. We have recently shown that the helical twisting of rhm1 petals results from decreased levels of rhamnose-containing cell wall polymers. In this work, we find that flavonols indirectly modify the rhm1 helical petal phenotype by altering rhamnose flux to the cell wall. Given this finding, we further investigated the relationship between flavonols and the cell wall in rhm1 cotyledons. We show that decreased flavonol rhamnosylation is not responsible for the cotyledon phenotype of rhm1 mutants. Instead, flavonols provide a large reservoir of rhamnose, and blocking flavonol synthesis or rhamnosylation can suppress rhm1 defects by diverting rhamnose to the synthesis of cell wall polysaccharides. Therefore, rhamnose is required in the cell wall for normal expansion of cotyledon epidermal cells. Our findings suggest a broad role for rhamnose-containing cell wall polysaccharides in the morphogenesis of epidermal cells.

plant biology↗

bioRxiv2017

A stable beneficial symbiotic relationship between endophytic fungus Schizophyllum commune and host plant Panax ginseng

Endophytes and plants can establish specific long-term symbiosis through the accumulation of secondary metabolites. Interactions between microbial inhabitants represent a novel area of study for natural products research. In this study, a strain of endophyte 3R-2 that can enhance the biomass and contents of ginsenoside Rc, ginsenoside Rg2 and ginsenoside Rg3 of Panax ginseng hairy roots was screened out via HPLC, which was identified as Schizophyllum commune through the morphological and molecular identification. On the base, we found the infection of the endophyte were obviously observed widely in the P. ginseng and the strain formed a stable relationship with P. ginseng hairy roots in parenchyma cells around through tissues embedding slicing, HE ammonium silver staining and immunofluorescence staining. On the other hand, elicitors of fungus 3R-2 can also significantly promote hairy root growth and contents of several ginsenosides, even several times higher than 3R-2 mycelium did. Moreover, S. commune 3R-2 mycelium and its elicitor could enhance the transcriptional activity of key genes during the ginsenosides biosynthetic pathway dramatically. Thus, endophyte S. commune 3R-2 and its elicitor change the chemical substance content by regulating the expression of genes involved in the secondary metabolite biosynthetic pathway.

plant biology↗

bioRxiv2017

Whole Root Transcriptomic Analysis Reveals a Role for Auxin Pathways in Resistance to Ralstonia solanacearum in Tomato

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt, and causes significant crop loss in the Solanaceae family. The pathogen first infects roots, which are a critical source of resistance in tomato (Solanum lycopersicum L.). Roots of both resistant and susceptible plants are colonized by the pathogen, yet rootstocks can provide significant levels of resistance. Currently, mechanisms of this root-mediated resistance remain largely unknown. To identify the molecular basis of this resistance, we analyzed the genome-wide transcriptional response of roots of resistant (Hawaii 7996) and susceptible (West Virginia700) tomatoes at multiple time points after inoculation with R. solanacearum. We found that defense pathways in roots of the resistant Hawaii7996 are activated earlier and more strongly than roots of susceptible West Virginia700. Further, auxin signaling and transport pathways are suppressed in roots of the resistant variety. Functional analysis of an auxin transport mutant in tomato confirmed a role for auxin pathways in bacterial wilt. Together, our results suggest that roots mediate resistance to R. solanacearum through genome-wide transcriptomic changes that result in strong activation of defense genes and alteration of auxin pathways.

plant biology↗

bioRxiv2017

Regulatory divergence of flowering time genes in the allopolyploid Brassica napus

Polyploidy is a recurrent feature of eukaryotic evolution and has been linked to increases in complexity, adaptive radiation and speciation. Within angiosperms, such events occur repeatedly in many plant lineages. We investigated the role of duplicated genes in the regulation of flowering in Brassica napus. This relatively young allotetraploid represents a snapshot of evolution and artificial selection in progress. In line with the gene balance hypothesis, we find preferential retention of expressed flowering time genes relative to the whole genome. Furthermore, gene expression dynamics across development reveal diverged regulation of many flowering time gene copies. This finding supports the concept of responsive backup circuits being key for the retention of duplicated genes. A case study of BnaTFL1 reveals differences in cis-regulatory elements downstream of these genes that could explain this divergence. Such differences in the regulatory dynamics of duplicated genes highlight the challenges for translating gene networks from model to more complex polyploid crop species.

plant biology↗

bioRxiv2017

The impact of dew-induced transpiration suppression on the water and isotope balances of Colocasia esculenta leaves

Foliar uptake of water from the surface of leaves is common when rainfall is scarce and non-meteoric water such as dew or fog is more abundant. However, many species in more mesic environments have hydrophobic leaves that do not allow the plant to uptake water. Unlike foliar uptake, all species can benefit from dew- or fog-induced transpiration suppression, but despite its ubiquity, transpiration suppression has so far never been quantified. Here, we investigate the effect of dew-induced transpiration suppression on the water balance and the isotope composition of leaves via a series of experiments. Characteristically hydrophobic leaves of a tropical plant, Colocasia esculenta, are misted with isotopically enriched water to reproduce dew deposition. This species does not uptake water from the surface of its leaves. We measure leaf water isotopes and water potential and find that misted leaves exhibit a higher water potential (p < 0.05) and a more depleted water isotope composition than dry leaves (p < 0.001), suggesting a ~30% decrease in transpiration rate (p < 0.001) compared to control leaves. We propose three possible mechanisms governing the interaction of water droplets with leaf energy balance: increase in albedo from the presence of dew droplets, decrease in leaf temperature from the evaporation of dew, and local decrease in vapor pressure deficit. Comparing previous studies on foliar uptake to our results, we conclude that transpiration suppression has an effect of similar amplitude, yet opposite sign to foliar uptake on leaf water isotopes.

plant biology↗

bioRxiv2017

Limited role of differential fractionation in genome content variation and function in maize (Zea mays L.) inbred lines

Maize is a diverse paleotetraploid species with widespread presence/absence variation and copy number variation. One mechanism through which presence/absence variation can arise is differential fractionation. Fractionation refers to the loss of duplicate gene pairs from one of the maize subgenomes during diploidization and differential fractionation refers to non-shared gene loss events between individuals. We investigated the prevalence of presence/absence variation resulting from differential fractionation in the syntenic portion of the genome using two whole genome de novo assemblies of the inbred lines B73 and PH207. Between these two genomes, syntenic genes were highly conserved with less than 1% of syntenic genes being subject to differential fractionation. The few variable syntenic genes that were identified are unlikely to contribute to functional phenotypic variation, as there is a significant depletion of these genes in annotated gene sets. In further comparisons of 60 diverse inbred lines, non-syntenic genes were six times more likely to be variable compared to syntenic genes, suggesting that comparisons among additional genome assemblies are not likely to result in the discovery of large-scale presence/absence variation among syntenic genes.\n\nSIGNIFICANCE STATEMENTThere is a large amount of presence/absence variation for gene content in maize. One mechanism that has been hypothesized to contribute to this variation is differential fractionation between individuals following the maize whole genome duplication event. Using comparative genomics, with sorghum and rice representing the ancestral state, we observed little evidence of differential fractionation among elite inbred lines and the few differentially fractionated genes identified did not appear to confer functional significance.

plant biology↗

bioRxiv2017

Nanoparticle-Guided Biomolecule Delivery for Transgene Expression and Gene Silencing in Mature Plants

Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis, and agricultural crop engineering. The plant cell wall is a barrier that limits the ease and throughput with which exogenous biomolecules can be delivered to plants. Current delivery methods either suffer from host range limitations, low transformation efficiencies, tissue damage, or unavoidable DNA integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into tissues and organs of intact plants of several species with a suite of pristine and chemically-functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We also demonstrate a second nanoparticle-based strategy in which small interfering RNA (siRNA) is delivered to Nb leaves and silence a gene with 95% efficiency. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene integration, into plant cells for diverse biotechnology applications.

plant biology↗

bioRxiv2017

NINJA-Associated ERF19 Negatively Regulates Arabidopsis Pattern-Triggered Immunity

Recognition of microbe-associated molecular patterns (MAMPs) derived from invading pathogens by plant pattern recognition receptors (PRRs) initiates defense responses known as pattern-triggered immunity (PTI). Transcription factors (TFs) orchestrate the onset of PTI through complex signaling networks. Here, we characterize the function of ERF19, a member of the Arabidopsis thaliana ethylene response factor (ERF) family. ERF19 was found to act as a negative regulator of PTI against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 (Pst). Notably, overexpression of ERF19 increased plant susceptibility to these pathogens and repressed MAMP-induced PTI outputs. In contrast, expression of the chimeric dominant repressor ERF19-SRDX boosted PTI activation, conferred increased resistance to B. cinerea, and enhanced elf18-triggered immunity against Pst. Consistent with a negative role of ERF19 in PTI, MAMP-mediated growth inhibition was respectively weakened or augmented in lines overexpressing ERF19 or expressing ERF19-SRDX. Moreover, we demonstrate that the transcriptional repressor Novel INteractor of JAZ (NINJA) associates with and represses the function of ERF19. Our work reveals ERF19 as a key player in a buffering mechanism to avoid defects imposed by over-activation of PTI and a potential role for NINJA in fine-tuning ERF19-mediated regulation.

plant biology↗

bioRxiv2017

A plant receptor-like kinase promotes cell-to-cell spread of RNAi and is targeted by a virus

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an anti-viral immune response. How this cell-to-cell spread of silencing is regulated is currently unknown. Here, we describe that the C4 protein from Tomato yellow leaf curl virus has the ability to inhibit the intercellular spread of RNAi. Using this viral protein as a probe, we have identified the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) as a positive regulator of the cell-to-cell movement of RNAi, and determined that BAM1 and its closest homologue, BAM2, play a redundant role in this process. C4 interacts with the intracellular domain of BAM1 and BAM2 at the plasma membrane and plasmodesmata, the cytoplasmic connections between plant cells, interfering with the function of these RLKs in the cell-to-cell spread of RNAi. Our results identify BAM1 as an element required for the cell-to-cell spread of RNAi and highlight that signalling components have been co-opted to play multiple functions in plants.

plant biology↗

bioRxiv2017

MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs

First paragraphDodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bi-directional movement of viruses, proteins, and mRNAs between host and parasite1, but the functional effects of these movements are not clear. Here we show that C. campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana hosts. Many of these miRNAs are 22 nts long, a usually rare size of plant miRNA associated with amplification of target silencing through secondary small interfering RNA (siRNA) production2. Several A. thaliana mRNAs are targeted by C. campestris 22 nt miRNAs during parasitism, resulting in mRNA cleavage, secondary siRNA production, and decreased mRNA accumulation levels. Hosts with mutations in two of the targets supported significantly higher growth of C. campestris. Homologs of target mRNAs from diverse plants also have predicted target sites to induced C. campestris miRNAs, and the same miRNAs are expressed and active against host targets when C. campestris parasitizes a different host, Nicotiana benthamiana. These data show that C. campestris miRNAs act as trans-species regulators of host gene expression, and suggest that they may act as virulence factors during parasitism.

plant biology↗

bioRxiv2017

Nuclear transcriptomes at high resolution using retooled INTACT

Isolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here we describe transfer of the Isolation of Nuclei from TAgged specific Cell Types (INTACT) to the monocot rice (Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear envelope-targeting domain of the Nuclear Tagging Fusion (NTF) protein with an outer nuclear envelope-anchored domain. This modified NTF was combined with codon optimized E. coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping and profiling of the complete nuclear transcriptome, including a rRNA degradation procedure that minimizes pre-rRNA transcripts. A high-resolution comparison of nuclear and steady-state poly (A)+ transcript populations of seedling root tips confirmed the capture of pre-mRNA and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell-types of rice and other species.\n\nSummaryImproved technology and methodology for affinity purification of nuclei and analysis of nuclear transcriptomes, chromatin and other nuclear components.

plant biology↗