Viral suppressor of RNA silencing in vascular plants also interferes with the development of the bryophyte Physcomitrella patens.

Plant viruses are important pathogens able to overcome plant defense mechanisms using their viral suppressors of RNA silencing (VSR). Small RNA pathways of bryophytes and vascular plants have significant similarities, but little is known about how viruses interact with mosses. This study elucidated the responses of Physcomitrella patens to two different VSRs. We transformed P. patens plants to express VSR P19 from tomato bushy stunt virus and VSR 2b from cucumber mosaic virus, respectively. RNA sequencing and quantitative PCR were used to detect the effects of VSRs on gene expression. Small RNA (sRNA) sequencing was used to estimate the influences of VSRs on the sRNA pool of P. patens. Expression of either VSR-encoding gene caused developmental disorders in P. patens. The transcripts of four different transcription factors (AP2/erf, EREB-11 and two MYBs) accumulated in the P19 lines. sRNA sequencing revealed that VSR P19 significantly changed the microRNA pool in P. patens. Our results suggest that VSR P19 is functional in P. patens and affects the abundance of specific microRNAs interfering with gene expression. The results open new opportunities for using Physcomitrella as an alternative system to study plant-virus interactions.

First report of Soybean dwarf virus infecting white clover (Trifolium repens) in Finland.

Soybean dwarf virus (SbDV, genus Luteovirus) is a single-stranded positive-sense RNA virus able to infect several legume species. SbDV was first reported in Japan where it was associated with significant yield losses in soybean (Tamada, 1969). Since then the virus has been detected worldwide. In Europe, the virus has only been reported from Germany (Abraham et al. 2007; Gaafar et al. 2020). In July 2018, several white clover plants (Trifolium repens L.) with leaf discoloration were observed in different locations in Oulu region in northern Finland. Individual plants were collected and analysed for the presence of viruses using small-RNA (sRNA) sequencing (Kreuze et. al. 2009) and reverse transcription-PCR (RT-PCR). Total RNA was extracted using EZNA micro RNA kit (Omega Bio-Tek, GA, USA). For sRNA analysis, sequencing libraries were constructed using the TruSeq small RNA library prep kit (Illumina, CA, USA) and sequenced on Illumina MiSeq platform. On average, 1.3 million single-end reads were obtained per sample, of which 27% were 18-25 nt long and used for the subsequent analysis. Contig assembly and virus identification with VirusDetect software (Zheng et al. 2017) detected SbDV in five out of six white clover samples analysed. Depending on the sample, 26-39 contigs (with lengths up to 301-469 nt) aligned to complete genome of a SbDV isolate previously described from white clover in USA (accession no. JN674402). The cumulative alignment coverage ranged from 35.5 % to 65.3 % with nucleotide identities between 94.4 % and 97.3 %. Additionally, two samples seemed to contain an unidentified closterovirus and one contained White clover cryptic virus 2. No additional viruses were detected from two of the samples.To confirm the presence of SbDV, the samples were tested by RT-PCR using primers MDF, MYF and MUR in multiplex (Schneider et al. 2011) together with SuperScript III One-Step RT-PCR System with the Platinum Taq DNA polymerase kit (Thermo Fisher Scientific, USA), essentially as instructed by the manufacturer. RT-PCR product of approximately 400 bp was produced from each of the five samples previously tested SbDV positive by sRNA analysis. No products were produced from the sample that was SbDV negative in sRNA analysis. Direct sequencing of two of the PCR products produced 347 and 361 bp sequences (GenBank: MZ355392 and MW929169) that were 95.7 % and 95.2 % identical, respectively, to a SbDV isolate (accession no. AB038148) that causes yellowing on soybean and is transmitted by Acyrthosiphon pisum (Terauchi et al. 2003). To our knowledge this is the first report of SbDV in Finland. SbDV is transmitted only by aphids (neither mechanical nor seed transmission occurs). In siRNA analysis all the isolates from Finland formed contigs that aligned almost perfectly (100 % coverage with ≥ 99 % nucleotide identity) to the coat protein (accession no. EF466131) of an SbDV isolate transmittable from white clover to faba bean by A. pisum (Abraham et al. 2007), an aphid common in Finland. Although significant yield losses by SbDV have only been reported on soybean (Tamada, 1969), the virus also causes symptoms in other legume crops, such as growth reduction on pea (Tian et al. 2017) and faba bean (Abraham et al. 2007), both of which are cultivated in Finland. References: Abraham et al. 2007. Plant Dis. 91: 1059. Gaafar et al. 2020. Front microbiol. 11: 583242. Kreuze et al. 2009. Virology 388:1. Schneider et al. 2011. Virology 412: 46. Tamada. 1969. Ann Phytopathol Soc Jpn. 35: 282. Terauchi et al. 2003. Phytopathology 93: 1560. Tian et al. 2017. Viruses 9: 155. Zheng et al. 2017. Virology 500: 130.

Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-ß-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.

Dissecting Abscisic Acid Signaling Pathways Involved in Cuticle Formation.

The cuticle is the outer physical barrier of aerial plant surfaces and an important interaction point between plants and the environment. Many environmental stresses affect cuticle formation, yet the regulatory pathways involved remain undefined. We used a genetics and gene expression analysis in Arabidopsis thaliana to define an abscisic acid (ABA) signaling loop that positively regulates cuticle formation via the core ABA signaling pathway, including the PYR/PYL receptors, PP2C phosphatase, and SNF1-Related Protein Kinase (SnRK) 2.2/SnRK2.3/SnRK2.6. Downstream of the SnRK2 kinases, cuticle formation was not regulated by the ABA-responsive element-binding transcription factors but rather by DEWAX, MYB16, MYB94, and MYB96. Additionally, low air humidity increased cuticle formation independent of the core ABA pathway and cell death/reactive oxygen species signaling attenuated expression of cuticle-biosynthesis genes. In Physcomitrella patens, exogenous ABA suppressed expression of cuticle-related genes, whose Arabidopsis orthologs were ABA-induced. Hence, the mechanisms regulating cuticle formation are conserved but sophisticated in land plants. Signaling specifically related to cuticle deficiency was identified to play a major role in the adaptation of ABA signaling pathway mutants to increased humidity and in modulating their immunity to Botrytis cinerea in Arabidopsis. These results define a cuticle-specific downstream branch in the ABA signaling pathway that regulates responses to the external environment.

Expression of Toll-like receptors in nasal epithelium in allergic rhinitis.

Toll-like receptors (TLRs) are important in barrier homeostasis, but their role in airborne allergies is not fully understood. The aim was to evaluate baseline and allergen-induced expression of TLR proteins in nasal epithelium during allergic rhinitis. Nineteen otherwise healthy non-smoking volunteers both allergic to birch pollen and non-allergic controls were enrolled. We took nasal biopsies before and after off-seasonal intranasal birch pollen or diluent challenge. The expression of epithelial TLR1-7, TLR9-10, and MyD88 proteins was immunohistochemically evaluated from the nasal biopsies. The TLR1-3 and TLR5-10 mRNAs were observed by RNA-microarray. Baseline epithelial expression of TLR proteins was wide and identical in controls and atopics. After off-seasonal intranasal birch pollen challenge, a negative change in the expression score of TLR1 and TLR6 proteins was detected in the atopic group. TLR mRNA expression was not affected by birch pollen challenge. Nasal epithelium seems to express all known TLRs. The mechanisms by which TLR1, and TLR6 proteins could affect pollen allergen transport need further studies.

Physcomitrella patens has kinase-LRR R gene homologs and interacting proteins.

Plant disease resistance gene (R gene)-like sequences were screened from the Physcomitrella patens genome. We found 603 kinase-like, 475 Nucleotide Binding Site (NBS)-like and 8594 Leucine Rich Repeat (LRR)-like sequences by homology searching using the respective domains of PpC24 (Accession No. BAD38895), which is a candidate kinase-NBS-LRR (kinase-NL) type R-like gene, as a reference. The positions of these domains in the genome were compared and 17 kinase-NLs were predicted. We also found four TIR-NBS-LRR (TIR-NL) sequences with homology to Arabidopsis TIR-NL (NM_001125847), but three out of the four TIR-NLs had tetratricopeptide repeats or a zinc finger domain in their predicted C-terminus. We also searched for kinase-LRR (KLR) type sequences by homology with rice OsXa21 and Arabidopsis thaliana FLS2. As a result, 16 KLRs with similarity to OsXa21 were found. In phylogenetic analysis of these 16 KLRs, PpKLR36, PpKLR39, PpKLR40, and PpKLR43 formed a cluster with OsXa21. These four PpKLRs had deduced transmembrane domain sequences and expression of all four was confirmed. We also found 14 homologs of rice OsXB3, which is known to interact with OsXa21 and is involved in signal transduction. Protein-protein interaction was observed between the four PpKLRs and at least two of the XB3 homologs in Y2H analysis.

Protein secretome of moss plants (Physcomitrella patens) with emphasis on changes induced by a fungal elicitor.

Studies on extracellular proteins (ECPs) contribute to understanding of the multifunctional nature of apoplast. Unlike vascular plants (tracheophytes), little information about ECPs is available from nonvascular plants, such as mosses (bryophytes). In this study, moss plants (Physcomitrella patens) were grown in liquid culture and treated with chitosan, a water-soluble form of chitin that occurs in cell walls of fungi and insects and elicits pathogen defense in plants. ECPs released to the culture medium were compared between chitosan-treated and nontreated control cultures using quantitative mass spectrometry (Orbitrap) and 2-DE-LC-MS/MS. Over 400 secreted proteins were detected, of which 70% were homologous to ECPs reported in tracheophyte secretomes. Bioinformatics analyses using SignalP and SecretomeP predicted classical signal peptides for secretion (37%) or leaderless secretion (27%) for most ECPs of P. patens, but secretion of the remaining proteins (36%) could not be predicted using bioinformatics. Cultures treated with chitosan contained 72 proteins not found in untreated controls, whereas 27 proteins found in controls were not detected in chitosan-treated cultures. Pathogen defense-related proteins dominated in the secretome of P. patens, as reported in tracheophytes. These results advance knowledge on protein secretomes of plants by providing a comprehensive account of ECPs of a bryophyte.

Involvement of a class III peroxidase and the mitochondrial protein TSPO in oxidative burst upon treatment of moss plants with a fungal elicitor.

Production of apoplastic reactive oxygen species (ROS), or oxidative burst, is among the first responses of plants upon recognition of microorganisms. It requires peroxidase or NADPH oxidase (NOX) activity and factors maintaining cellular redox homeostasis. Here, PpTSPO1 involved in mitochondrial tetrapyrrole transport and abiotic (salt) stress tolerance was tested for its role in biotic stress in Physcomitrella patens, a nonvascular plant (moss). The fungal elicitor chitin caused an immediate oxidative burst in wild-type P. patens but not in the previously described ΔPrx34 mutants lacking the chitin-responsive secreted class III peroxidase (Prx34). Oxidative burst in P. patens was associated with induction of the oxidative stress-related genes AOX, LOX7, and NOX, and also PpTSPO1. The available ΔPpTSPO1 knockout mutants overexpressed AOX and LOX7 constitutively, produced 2.6-fold more ROS than wild-type P. patens, and exhibited increased sensitivity to a fungal necrotrophic pathogen and a saprophyte. These results indicate that Prx34, which is pivotal for antifungal resistance, catalyzes ROS production in P. patens, while PpTSPO1 controls redox homeostasis. The capacity of TSPO to bind harmful free heme and porphyrins and scavenge them through autophagy, as shown in Arabidopsis under abiotic stress, seems important to maintenance of the homeostasis required for efficient pathogen defense.

Infection of the Sunagoke moss panels with fungal pathogens hampers sustainable greening in urban environments.

Drought and heat tolerance of the Sunagoke moss (Racomitrium japonicum) and the low thermal conductivity of the dry moss tissue offer novel greening and insulation possibilities of roofs and walls to mitigate the heat island phenomenon in urban environments. However, damage may appear in the moss panels under humid conditions in Japan. In this study we characterized fungi associated with the damaged areas of the Sunagoke moss panels. Fungi were identified by morphology and internal transcribed spacer (ITS) sequence analysis and tested for pathogenicity on R. japonicum (Grimmiaceae) and an unrelated moss species (Physcomitrella patens; Funariaceae) under controlled conditions. Alternaria alternata, Fusarium avenaceum and Fusarium oxysporum caused severe necrosis and death, whereas Cladosporium oxysporum and Epicoccum nigrum caused milder discoloration or chlorosis in both moss species. The fungi pathogenic on moss were closely related to fungal pathogens described from cultivated vascular plants. Ammonium increased severity of fungal diseases in moss. This study demonstrated that fungi can cause economically significant diseases in cultivated moss and hamper commercial use of the moss panels unless appropriate control methods are developed. Use of a single moss clone to cover large surfaces and the air pollutants such as ammonium may increase the risk for fungal disease problems.

Indoleamine 2,3-dioxygenase expression in patients with allergic rhinitis: a case-control study.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is a tryptophan catalyzing enzyme. It has been suggested that it has a role in lower airway allergic inflammations, but its role in allergic rhinitis has not been investigated. OBJECTIVE: Our aim was to evaluate the expression of IDO in the nasal mucosa of allergic rhinitis patients allergic to birch pollen during peak exposure to birch pollen allergen and compare it to non-atopic patients. METHODS: IDO expression was immunohistochemically evaluated from nasal specimens obtained in- and off-season from otherwise healthy non-smoking volunteers both allergic to birch pollen (having mild or moderate allergic rhinoconjunctivitis) and non-allergic controls. RESULTS: The IDO expression levels were low in healthy controls and remained low also in patients allergic to birch pollen. There were no differences in the expression of IDO in- and off-season in either healthy or allergic subjects. CONCLUSIONS: There is a controversy in the role of IDO in upper and lower airways during allergic airway disease. It seems that IDO is associated to allergic inflammations of the lower airways, but does not have a local role in the nasal cavity at least in mild or moderate forms of allergic rhinitis.

Quickly-released peroxidase of moss in defense against fungal invaders.

Mosses (Bryophyta) are nonvascular plants that constitute a large part of the photosynthesizing biomass and carbon storage on Earth. Little is known about how this important portion of flora maintains its health status. This study assessed whether the moss, Physcomitrella patens, responds to treatment with chitosan, a fungal cell wall-derived compound inducing defense against fungal pathogens in vascular plants. Application of chitosan to liquid culture of P. patens caused a rapid increase in peroxidase activity in the medium. For identification of the peroxidase(s), matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/MS, other methods and the whole-genome sequence of P. patens were utilized. Peroxidase gene knock-out mutants were made and inoculated with fungi. The peroxidase activity resulted from a single secreted class III peroxidase (Prx34) which belonged to a P. patens specific phylogenetic cluster in analysis of the 45 putative class III peroxidases of P. patens and those of Arabidopsis and rice. Saprophytic and pathogenic fungi isolated from another moss killed the Prx34 knockout mutants but did not damage wild-type P. patens. The data point out the first specific host factor that is pivotal for pathogen defense in a nonvascular plant. Furthermore, results provide conclusive evidence that class III peroxidases in plants are needed in defense against hostile invasion by fungi.

Caveolar transport through nasal epithelium of birch pollen allergen Bet v 1 in allergic patients.

BACKGROUND: Previous work in type I pollen allergies has focused on aberrant immunoresponses. OBJECTIVE: Our systems-level analyses explore the role of epithelium in early pathogenesis of type I allergic reactions. METHODS: We began top-down analyses of differences in human nasal epithelial cells and biopsy specimens obtained from patients with birch allergy and healthy control subjects in the resting state and after intranasal in vivo birch pollen challenges. Immunohistochemistry, immunotransmission electron microscopy, mass spectrometry, transcriptomics, and integration of data to a pathway were conducted. RESULTS: Bet v 1 allergen bound to epithelium immediately after in vivo birch pollen challenge during winter only in allergic individuals. It also travelled through epithelium with caveolae to mast cells. Sixteen unique proteins were found to bind to the Bet v 1 column only in lysates from allergic epithelial cells; 6 of these were caveolar and 6 were cytoskeletal proteins. The nasal epithelial transcriptome analysis from allergic and healthy subjects differed during the winter season, and these subjects also responded differentially to birch pollen challenge. Within this pollen-induced response, the gene ontology categories of cytoskeleton and actin cytoskeleton were decreased in allergic patients, whereas the actin-binding category was enriched in healthy subjects. Integration of microscopic, mass spectrometric, and transcriptomic data to a common protein-protein binding network showed how these were connected to each other. CONCLUSION: We propose a hypothesis of caveolae-dependent uptake and transport of birch pollen allergen in the epithelium of allergic patients only. Application of discovery-driven methodologies can provide new hypotheses worth further analysis of complex multifactorial diseases, such as type I allergy.

Cloning, characterization and localization of three novel class III peroxidases in lignifying xylem of Norway spruce (Picea abies).

Plant class III peroxidases (POXs) take part in the formation of lignin and maturation of plant cell walls. However, only a few examples of such peroxidases from gymnosperm tree species with highly lignified xylem tracheids have been implicated so far. We report here cDNA cloning of three xylem-expressed class III peroxidase encoding genes from Norway spruce (Picea abies). The translated proteins, PX1, PX2 and PX3, contain the conserved amino acids required for heme-binding and peroxidase catalysis. They all begin with putative secretion signal propeptide sequences but diverge substantially at phylogenetic level, grouping to two subclusters when aligned with other class III plant peroxidases. In situ hybridization analysis on expression of the three POXs in Norway spruce seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm of young, developing tracheids within the current growth ring where lignification is occurring. Function of the putative N-terminal secretion signal peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions with EGFP (enhanced green fluorescent protein) and expressing them in tobacco protoplasts. Full-length coding region of px1 was also heterologously expressed in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1 peroxidase is processed via the endoplasmic reticulum (ER) most likely for secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal localization for participation in the maturation of the spruce tracheid secondary cell wall.

Developmental lignification and seasonal variation in beta-glucosidase and peroxidase activities in xylem of Scots pine, Norway spruce and silver birch.

We examined the relationship between beta-glucosidase and peroxidase activities and xylem lignification in the stems of Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies (L.) Karst.) and silver birch (Betula pendula Roth) during the 1999 growing season. Examination of stem cross sections stained with safranin and Alcian blue for lignin and cellulose, respectively, indicated that radial growth of pine and spruce xylem began in late May, whereas the growth of birch xylem was initiated 2 weeks later. Lignification began soon after thickening of the newly formed cell walls, i.e., upon deposition of cellulose. Hydrolysis of the synthetic beta-glucosidase substrate p-nitrophenyl-beta-O-D-glucopyranoside was correlated with radial growth and lignification in the xylem of both conifers, but the relationship between lignification and the hydrolysis of coniferin by beta-glucosidase was not obvious. Beta-glucosidase activities in the xylem of silver birch were low and did not correlate with growth or lignification with either substrate. An increase in peroxidase activity was detected at the initiation of growth and lignification in the conifers and during growth and lignification in silver birch, but high peroxidase activities were also measured outside the growth period during late autumn, winter and early spring.