Expression of major photosynthetic and salt-resistance genes in invasive reed lineages grown under elevated CO2 and temperature

It is important to investigate the molecular causes of the variation in ecologically important traits to fully understand phenotypic responses to climate change. In the Mississippi River Delta, two distinct, sympatric invasive lineages of common reed (Phragmites australis) are known to differ in several ecophysiological characteristics and are expected to become more salt resistant due to increasing atmospheric CO2 and temperature. We investigated whether different patterns of gene expression can explain their ecophysiological differences and increased vigor under future climatic conditions. We compared the transcript abundance of photosynthetic genes of the Calvin cycle (Rubisco small subunit, RbcS; Phosphoglycerate kinase, PGK; Phosphoribulokinase, PRK), genes related with salt transport (Na+/H+ antiporter, PhaNHA) and oxidative stress response genes (Manganese Superoxide dismutase, MnSOD; Glutathione peroxidase, GPX), and the total aboveground biomass production between two genotypes representing the two lineages. The two genotypes (Delta-type, Mediterranean lineage, and EU-type, Eurasian lineage) were grown under an ambient and a future climate scenario with simultaneously elevated CO2 and temperature, and under two different soil salinities (0‰ or 20‰). We found neither differences in the aboveground biomass production nor the transcript abundances of the two genotypes, but soil salinity significantly affected all the investigated parameters, often interacting with the climatic conditions. At 20‰ salinity, most genes were higher expressed in the future than in the ambient climatic conditions. Higher transcription of the genes suggests higher abundance of the protein they code for, and consequently increased photosynthate production, improved stress responses, and salt exclusion. Therefore, the higher expression of these genes most likely contributed to the significantly ameliorated salinity impact on the aboveground biomass production of both P. australis genotypes under elevated temperature and CO2. Although transcript abundances did not explain differences between the lineages, they correlated with the increased vigor of both lineages under anticipated future climatic conditions. Two sympatric, highly invasive lineages of Phragmites australis from North America differ in their ecophysiological responses to salinity and climate, and differences in gene expression may cause these different phenotypic traits. Under a future climatic scenario and high soil salinity, the expression of photosynthetic and salt-stress related genes was increased in both lineages, relative to the ambient climate, but the phenotypic differences between the lineages were not explained by the gene expression. Similar changes in gene expression may therefore facilitate salt resistance and an increased invasive vigour of both reed lineages under the changing climate

LYS12 LysM receptor decelerates Phytophthora palmivora disease progression in Lotus japonicus.

Phytophthora palmivora is a devastating oomycete plant pathogen. We found that P. palmivora induces disease in Lotus japonicus and used this interaction to identify cellular and molecular events in response to this oomycete, which has a broad host range. Transcript quantification revealed that Lys12 was highly and rapidly induced during P. palmivora infection. Mutants of Lys12 displayed accelerated disease progression, earlier plant death and a lower level of defence gene expression than the wild type, while the defence program after chitin, laminarin, oligogalacturonide or flg22 treatment and the root symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhiza were similar to the wild type. On the microbial side, we found that P. palmivora encodes an active chitin synthase-like protein, and mycelial growth is impaired after treatment with a chitin-synthase inhibitor. However, wheat germ agglutinin-detectable N-acetyl-glucosamine (GlcNAc) epitopes were not identified when the oomycete was grown in vitro or while infecting the roots. This indicates that conventional GlcNAc-mers are unlikely to be produced and/or accumulate in P. palmivora cell walls and that LYS12 might perceive an unknown carbohydrate. The impact of Lys12 on progression of root rot disease, together with the finding that similar genes are present in other P. palmivora hosts, suggests that LYS12 might mediate a common early response to this pathogen

Application of ecosystem-specific reference databases for increased taxonomic resolution in soil microbial profiling

Intensive agriculture systems have paved the way for a growing human population. However, the abundant use of mineral fertilizers and pesticides may negatively impact nutrient cycles and biodiversity. One potential alternative is to harness beneficial relationships between plants and plant-associated rhizobacteria to increase nutrient-use efficiency and provide pathogen resistance. Plant-associated microbiota profiling can be achieved using high-throughput 16S rRNA gene amplicon sequencing. However, interrogation of these data is limited by confident taxonomic classifications at high taxonomic resolution (genus- or species level) with the commonly applied universal reference databases. High-throughput full-length 16S rRNA gene sequencing combined with automated taxonomy assignment (AutoTax) can be used to create amplicon sequence variant resolved ecosystems-specific reference databases that are superior to the traditional universal reference databases. This approach was used here to create a custom reference database for bacteria and archaea based on 987,353 full-length 16S rRNA genes from Askov and Cologne soils. We evaluated the performance of the database using short-read amplicon data and found that it resulted in the increased genus- and species-level classification compared to commonly use universal reference databases. The custom database was utilized to evaluate the ecosystem-specific primer bias and taxonomic resolution of amplicon primers targeting the V5–V7 region of the 16S rRNA gene commonly used within the plant microbiome field. Finally, we demonstrate the benefits of custom ecosystem-specific databases through the analysis of V5–V7 amplicon data to identify new plant-associated microbes for two legumes and two cereal species

Integrative transcript to proteome analysis of barley during Ramularia collo-cygni leaf spot development identified several proteins that are related to fungal recognition and infection responses

IntroductionRamularia leaf spot (RLS) disease is a growing threat to barley cultivation, but with no substantial resistance identified to date. Similarly, the understanding of the lifestyle of Ramularia collo-cygni (Rcc) and the prediction of RLS outbreak severity remain challenging, with Rcc displaying a rather untypical long endophytic phase and a sudden change to a necrotrophic lifestyle. The aim of this study was to provide further insights into the defense dynamics during the different stages of colonization and infection in barley in order to identify potential targets for resistance breeding.MethodsUtilizing the strength of proteomics in understanding plant–pathogen interactions, we performed an integrative analysis of a published transcriptome dataset with a parallel generated proteome dataset. Therefore, we included two spring barley cultivars with contrasting susceptibilities to Rcc and two fungal isolates causing different levels of RLS symptoms.ResultsInterestingly, early responses in the pathogen recognition phase of the host were driven by strong responses differing between isolates. An important enzyme in this process is a xylanase inhibitor, which protected the plant from cell wall degradation by the fungal xylanase. At later time points, the differences were driven by cultivar-specific responses, affecting mostly features contributing to the pathogenesis- and senescence-related pathways or photosynthesis.DiscussionThis supports the hypothesis of a hemibiotrophic lifestyle of Rcc, with slight differences in trophism of the two analyzed isolates. The integration of these data modalities highlights a strength of protein-level analysis in understanding plant–pathogen interactions and reveals new features involved in fungal recognition and susceptibility in barley cultivars

Characterising standard genetic parts and establishing common principles for engineering legume and cereal roots

Plant synthetic biology and cereal engineering depends on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well-established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimisation, intron-mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small-scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multi-genic traits where the expression of each transgene needs to be precisely regulated. Here we present data on the characterisation of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimisation and intron-mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimisation and intron-mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use, and also propose a general set of principles and guidelines for those engineering cereal species

A combination of chitooligosaccharide and lipochitooligosaccharide recognition promotes arbuscular mycorrhizal associations in Medicago truncatula.

Plants associate with beneficial arbuscular mycorrhizal fungi facilitating nutrient acquisition. Arbuscular mycorrhizal fungi produce chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs), that promote symbiosis signalling with resultant oscillations in nuclear-associated calcium. The activation of symbiosis signalling must be balanced with activation of immunity signalling, which in fungal interactions is promoted by COs resulting from the chitinaceous fungal cell wall. Here we demonstrate that COs ranging from CO4-CO8 can induce symbiosis signalling in Medicago truncatula. CO perception is a function of the receptor-like kinases MtCERK1 and LYR4, that activate both immunity and symbiosis signalling. A combination of LCOs and COs act synergistically to enhance symbiosis signalling and suppress immunity signalling and receptors involved in both CO and LCO perception are necessary for mycorrhizal establishment. We conclude that LCOs, when present in a mix with COs, drive a symbiotic outcome and this mix of signals is essential for arbuscular mycorrhizal establishment

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5

Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities

Lotus japonicus has been used for decades as a model legume to study the establishment of binary symbiotic relationships with nitrogen-fixing rhizobia that trigger root nodule organogenesis for bacterial accommodation. Using community profiling of 16S rRNA gene amplicons, we reveal that in Lotus, distinctive noduleand root-inhabiting communities are established by parallel, rather than consecutive, selection of bacteria from the rhizosphere and root compartments. Comparative analyses of wild-type (WT) and symbiotic mutants in Nod factor receptor5 (nfr5), Nodule inception (nin) and Lotus histidine kinase1 (lhk1) genes identified a previously unsuspected role of the nodulation pathway in the establishment of different bacterial assemblages in the root and rhizosphere. We found that the loss of nitrogen-fixing symbiosis dramatically alters community structure in the latter two compartments, affecting at least 14 bacterial orders. The differential plant growth phenotypes seen between WT and the symbiotic mutants in nonsupplemented soil were retained under nitrogen-supplemented conditions that blocked the formation of functional nodules in WT, whereas the symbiosis-impaired mutants maintain an altered community structure in the nitrogen-supplemented soil. This finding provides strong evidence that the root-associated community shift in the symbiotic mutants is a direct consequence of the disabled symbiosis pathway rather than an indirect effect resulting from abolished symbiotic nitrogen fixation. Our findings imply a role of the legume host in selecting a broad taxonomic range of root-associated bacteria that, in addition to rhizobia, likely contribute to plant growth and ecological performance