Global expression patterns of R-genes in tomato and potato.

Introduction: As key-players of plant immunity, the proteins encoded by resistance genes (R-genes) recognize pathogens and initiate pathogen-specific defense responses. The expression of some R-genes carry fitness costs and therefore inducible immune responses are likely advantageous. To what degree inducible resistance driven by R-genes is triggered by pathogen infection is currently an open question. Methods: In this study we analyzed the expression of 940 R-genes of tomato and potato across 315 transcriptome libraries to investigate how interspecific interactions with microbes influence R-gene expression in plants. Results: We found that most R-genes are expressed at a low level. A small subset of R-genes had moderate to high levels of expression and were expressed across many independent libraries, irrespective of infection status. These R-genes include members of the class of genes called NRCs (NLR required for cell death). Approximately 10% of all R-genes were differentially expressed during infection and this included both up- and down-regulation. One factor associated with the large differences in R-gene expression was host tissue, reflecting a considerable degree of tissue-specific transcriptional regulation of this class of genes. Discussion: These results call into question the widespread view that R-gene expression is induced upon pathogen attack. Instead, a small core set of R-genes is constitutively expressed, imparting upon the plant a ready-to-detect and defend status.

What Drives the Assembly of Plant-associated Protist Microbiomes? Investigating the Effects of Crop Species, Soil Type and Bacterial Microbiomes.

In a field experiment we investigated the influence of the environmental filters soil type (i.e. three contrasting soils) and plant species (i.e. lettuce and potato) identity on rhizosphere community assembly of Cercozoa, a dominant group of mostly bacterivorous soil protists. Plant species (14%) and rhizosphere origin (vs bulk soil) with 13%, together explained four times more variation in cercozoan beta diversity than the three soil types (7% explained variation). Our results clearly confirm the existence of plant species-specific protist communities. Network analyses of bacteria-Cercozoa rhizosphere communities identified scale-free small world topologies, indicating mechanisms of self-organization. While the assembly of rhizosphere bacterial communities is bottom-up controlled through the resource supply from root (secondary) metabolites, our results support the hypothesis that the net effect may depend on the strength of top-down control by protist grazers. Since grazing of protists has a strong impact on the composition and functioning of bacteria communities, protists expand the repertoire of plant genes by functional traits, and should be considered as 'protist microbiomes' in analogy to 'bacterial microbiomes'.

Multiplex Real-Time Reverse Transcription PCR for Influenza A Virus, Influenza B Virus, and Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019, and the outbreak rapidly evolved into the current coronavirus disease pandemic. SARS-CoV-2 is a respiratory virus that causes symptoms similar to those caused by influenza A and B viruses. On July 2, 2020, the US Food and Drug Administration granted emergency use authorization for in vitro diagnostic use of the Influenza SARS-CoV-2 Multiplex Assay. This assay detects influenza A virus at 102.0, influenza B virus at 102.2, and SARS-CoV-2 at 100.3 50% tissue culture or egg infectious dose, or as few as 5 RNA copies/reaction. The simultaneous detection and differentiation of these 3 major pathogens increases overall testing capacity, conserves resources, identifies co-infections, and enables efficient surveillance of influenza viruses and SARS-CoV-2.

Inducible biosynthesis and immune function of the systemic acquired resistance inducer N-hydroxypipecolic acid in monocotyledonous and dicotyledonous plants.

Recent work has provided evidence for the occurrence of N-hydroxypipecolic acid (NHP) in Arabidopsis thaliana, characterized its pathogen-inducible biosynthesis by a three-step metabolic sequence from l-lysine, and established a central role for NHP in the regulation of systemic acquired resistance. Here, we show that NHP is biosynthesized in several other plant species in response to microbial attack, generally together with its direct metabolic precursor pipecolic acid and the phenolic immune signal salicylic acid. For example, NHP accumulates locally in inoculated leaves and systemically in distant leaves of cucumber in response to Pseudomonas syringae attack, in Pseudomonas-challenged tobacco and soybean leaves, in tomato inoculated with the oomycete Phytophthora infestans, in leaves of the monocot Brachypodium distachyon infected with bacterial (Xanthomonas translucens) and fungal (Magnaporthe oryzae) pathogens, and in M. oryzae-inoculated barley. Notably, resistance assays indicate that NHP acts as a potent inducer of acquired resistance to bacterial and fungal infection in distinct monocotyledonous and dicotyledonous species. Pronounced systemic accumulation of NHP in leaf phloem sap of locally inoculated cucumber supports a function for NHP as a phloem-mobile immune signal. Our study thus generalizes the existence and function of an NHP resistance pathway in plant systemic acquired resistance.

Rapid evolution in plant-microbe interactions - an evolutionary genomics perspective.

Access to greater genomic resolution through new sequencing technologies is transforming the field of plant pathology. As scientists embrace these new methods, some overarching patterns and observations come into focus. Evolutionary genomic studies are used to determine not only the origins of pathogen lineages and geographic patterns of genetic diversity, but also to discern how natural selection structures genetic variation across the genome. With greater and greater resolution, we can now pinpoint the targets of selection on a large scale. At multiple levels, crypsis and convergent evolution are evident. Host jumps and shifts may be more pervasive than once believed, and hybridization and horizontal gene transfer (HGT) likely play important roles in the emergence of genetic novelty.

Site-specific distribution of oak rhizosphere-associated oomycetes revealed by cytochrome c oxidase subunit II metabarcoding.

The phylum Oomycota comprises important tree pathogens like Phytophthora quercina, involved in central European oak decline, and Phytophthora cinnamomi shown to affect holm oaks among many other hosts. Despite the importance to study the distribution, dispersal and niche partitioning of this phylum, metabarcoding surveys, and studies considering environmental factors that could explain oomycete community patterns are still rare. We investigated oomycetes in the rhizosphere of evergreen oaks in a Spanish oak woodland using metabarcoding based on Illumina sequencing of the taxonomic marker cytochrome c oxidase subunit II (cox2). We developed an approach amplifying a 333 bp long fragment using the forward primer Hud-F (Mycologia, 2000) and a reverse primer found using DegePrime (Applied and Environmental Microbiology, 2014). Factors reflecting topo-edaphic conditions and tree health were linked to oomycete community patterns. The majority of detected OTUs belonged to the Peronosporales. Most taxa were relatives of the Pythiaceae, but relatives of the Peronosporaceae and members of the Saprolegniales were also found. The most abundant OTUs were related to Globisporangium irregulare and P. cinnamomi, both displaying strong site-specific patterns. Oomycete communities were strongly correlated with the environmental factors: altitude, crown foliation, slope and soil skeleton and soil nitrogen. Our findings illustrate the significance of small scale variation in habitat conditions for the distribution of oomycetes and highlight the importance to study oomycete communities in relation to such ecological patterns.

The evolutionary history of LysM-RLKs (LYKs/LYRs) in wild tomatoes.

BACKGROUND: The LysM receptor-like kinases (LysM-RLKs) are important to both plant defense and symbiosis. Previous studies described three clades of LysM-RLKs: LysM-I/LYKs (10+ exons per gene and containing conserved kinase residues), LysM-II/LYRs (1-5 exons per gene, lacking conserved kinase residues), and LysM-III (two exons per gene, with a kinase unlike other LysM-RLK kinases and restricted to legumes). LysM-II gene products are presumably not functional as conventional receptor kinases, but several are known to operate in complexes with other LysM-RLKs. One aim of our study was to take advantage of recently mapped wild tomato transcriptomes to evaluate the evolutionary history of LysM-RLKs within and between species. The second aim was to place these results into a broader phylogenetic context by integrating them into a sequence analysis of LysM-RLKs from other functionally well-characterized model plant species. Furthermore, we sought to assess whether the Group III LysM-RLKs were restricted to the legumes or found more broadly across Angiosperms. RESULTS: Purifying selection was found to be the prevailing form of natural selection within species at LysM-RLKs. No signatures of balancing selection were found in species-wide samples of two wild tomato species. Most genes showed a greater extent of purifying selection in their intracellular domains, with the exception of SlLYK3 which showed strong purifying selection in both the extracellular and intracellular domains in wild tomato species. The phylogenetic analysis did not reveal a clustering of microbe/functional specificity to groups of closely related proteins. We also discovered new putative LysM-III genes in a range of Rosid species, including Eucalyptus grandis. CONCLUSIONS: The LysM-III genes likely originated before the divergence of E. grandis from other Rosids via a fusion of a Group II LysM triplet and a kinase from another RLK family. SlLYK3 emerges as an especially interesting candidate for further study due to the high protein sequence conservation within species, its position in a clade of LysM-RLKs with distinct LysM domains, and its close evolutionary relationship with LYK3 from Arabidopsis thaliana.

The Elaboration of miRNA Regulation and Gene Regulatory Networks in Plant⁻Microbe Interactions.

: Plants are exposed to diverse abiotic and biotic stimuli. These require fast and specific integrated responses. Such responses are coordinated at the protein and transcript levels and are incorporated into larger regulatory networks. Here, we focus on the evolution of transcriptional regulatory networks involved in plant-pathogen interactions. We discuss the evolution of regulatory networks and their role in fine-tuning plant defense responses. Based on the observation that many of the cornerstones of immune signaling in angiosperms are also present in streptophyte algae, it is likely that some regulatory components also predate the origin of land plants. The degree of functional conservation of many of these ancient components has not been elucidated. However, ongoing functional analyses in bryophytes show that some components are conserved. Hence, some of these regulatory components and how they are wired may also trace back to the last common ancestor of land plants or earlier. Of course, an understanding of the similarities and differences during the evolution of plant defense networks cannot ignore the lineage-specific coevolution between plants and their pathogens. In this review, we specifically focus on the small RNA regulatory networks involved in fine-tuning of the strength and timing of defense responses and highlight examples of pathogen exploitation of the host RNA silencing system. These examples illustrate well how pathogens frequently target gene regulation and thereby alter immune responses on a larger scale. That this is effective is demonstrated by the diversity of pathogens from distinct kingdoms capable of manipulating the same gene regulatory networks, such as the RNA silencing machinery.

Small RNA molecules and their role in plant disease.

All plant species are subject to disease. Plant diseases are caused by parasites, e.g. viruses, bacteria, oomycetes, parasitic plants, fungi, or nematodes. In all organisms, gene expression is tightly regulated and underpins essential functions and physiology. The coordination and regulation of both host and pathogen gene expression is essential for pathogens to infect and cause disease. One mode of gene regulation is RNA silencing. This biological process is widespread in the natural world, present in plants, animals and several pathogens. In RNA silencing, small (20-40 nucleotides) non-coding RNAs (small-RNAs, sRNAs) accumulate and regulate gene expression transcriptionally or post-transcriptionally in a sequence-specific manner. Regulation of sRNA molecules provides a fast mode to alter gene activity of multiple gene transcripts. RNA silencing is an ancient mechanism that protects the most sensitive part of an organism: its genetic code. sRNA molecules emerged as regulators of plant development, growth and plant immunity. sRNA based RNA silencing functions both within and between organisms. Here we present the described sRNAs from plants and pathogens and discuss how they regulate host immunity and pathogen virulence. We speculate on how sRNA molecules can be exploited to develop disease resistant plants. Finally, the activity of sRNA molecules can be prevented by proteins that suppress RNA silencing. This counter silencing response completes the dialog between plants and pathogens controlling plant disease or resistance outcome on the RNA (controlling gene expression) and protein level.

Efficacy of biological agents and compost on growth and resistance of tomatoes to late blight.

MAIN CONCLUSION: This study identified biocontrol measures for improving plant quality and resistance under biotic stress caused by the most devastating pathogen in tomato production. The management of plant diseases are dependent on a variety of factors. Two important variables are the soil quality and its bacterial/fungal community. However, the interaction of these factors is not well understood and remains problematic in producing healthy crops. Here, the effect of oak-bark compost, Bacillus subtilis subsp. subtilis, Trichoderma harzianum and two commercial products (FZB24 and FZB42) were investigated on tomato growth, production of metabolites and resistance under biotic stress condition (infection with Phytophthora infestans). Oak-bark compost, B. subtilis subsp. subtilis, and T. harzianum significantly enhanced plant growth and immunity when exposed to P. infestans. However, the commercial products were not as effective in promoting growth, with FZB42 having the weakest protection. Furthermore, elevated levels of anthocyanins did not correlate with enhanced plant resistance. Overall, the most effective and consistent plant protection was obtained when B. subtilis subsp. subtilis was combined with oak-bark compost. In contrast, the combination of T. harzianum and oak-bark compost resulted in increased disease severity. The use of compost in combination with bio-agents should, therefore, be evaluated carefully for a reliable and consistent tomato protection.

On plant defense signaling networks and early land plant evolution.

All land plants must cope with phytopathogens. Algae face pathogens, too, and it is reasonable to assume that some of the strategies for dealing with pathogens evolved prior to the origin of embryophytes - plant terrestrialization simply changed the nature of the plant-pathogen interactions. Here we highlight that many potential components of the angiosperm defense toolkit are i) found in streptophyte algae and non-flowering embryophytes and ii) might be used in non-flowering plant defense as inferred from published experimental data. Nonetheless, the common signaling networks governing these defense responses appear to have become more intricate during embryophyte evolution. This includes the evolution of the antagonistic signaling pathways of jasmonic and salicylic acid, multiple independent expansions of resistance genes, and the evolution of resistance gene-regulating microRNAs. Future comparative studies will illuminate which modules of the streptophyte defense signaling network constitute the core and which constitute lineage- and/or environment-specific (peripheral) signaling circuits.

Expression profiling across wild and cultivated tomatoes supports the relevance of early miR482/2118 suppression for Phytophthora resistance.

Plants possess a battery of specific pathogen resistance (R-)genes. Precise R-gene regulation is important in the presence and absence of a pathogen. Recently, a microRNA family, miR482/2118, was shown to regulate the expression of a major class of R-genes, nucleotide-binding site leucine-rich repeats (NBS-LRRs). Furthermore, RNA silencing suppressor proteins, secreted by pathogens, prevent the accumulation of miR482/2118, leading to an upregulation of R-genes. Despite this transcriptional release of R-genes, RNA silencing suppressors positively contribute to the virulence of some pathogens. To investigate this paradox, we analysed how the regulation of NBS-LRRs by miR482/2118 has been shaped by the coevolution between Phytophthora infestans and cultivated and wild tomatoes. We used degradome analyses and qRT-PCR to evaluate and quantify the co-expression of miR482/2118 and their NBS-LRR targets. Our data show that miR482/2118-mediated targeting contributes to the regulation of NBS-LRRs in Solanum lycopersicum. Based on miR482/2118 expression profiling in two additional tomato species-with different coevolutionary histories with P. infestans-we hypothesize that pathogen-mediated RNA silencing suppression is most effective in the interaction between S. lycopersicum and P. infestans Furthermore, an upregulation of miR482/2118 early in the infection may increase susceptibility to P. infestans.

Broad-spectrum inhibition of Phytophthora infestans by fungal endophytes.

Phytophthora infestans is a devastating pathogen of tomato and potato. It readily overcomes resistance genes and applied agrochemicals and hence even today causes large yield losses. Fungal endophytes provide a largely unexplored avenue of control of Phy. infestans. Not only do endophytes produce a wide array of bioactive metabolites, they may also directly compete with and defeat pathogens in planta. Here, we tested 12 fungal endophytes isolated from different plant species in vitro for their production of metabolites with anti- Phytophthora activity. Four well-performing isolates were evaluated for their ability to suppress nine isolates of Phy. infestans on agar medium and in planta. Two endophytes reliably inhibited all Phy. infestans isolates on agar medium, of which Phoma eupatorii isolate 8082 was the most promising. It nearly abolished infection by Phy. infestans in planta. Our data indicate a role for the production of anti-Phytophthora compounds by the fungus and/or an enhanced plant defense response, as evident by an enhanced anthocyanin production. Here, we present a potential biocontrol agent, which can inhibit a broad-spectrum of Phy. infestans isolates. Such broadly acting inhibition is ideal, because it allows for effective control of genetically diverse isolates and may slow the adaptation of Phy. infestans.

Jasmonic and salicylic acid response in the fern Azolla filiculoides and its cyanobiont.

Plants sense and respond to microbes utilizing a multilayered signalling cascade. In seed plants, the phytohormones jasmonic and salicylic acid (JA and SA) are key denominators of how plants respond to certain microbes. Their interplay is especially well-known for tipping the scales in plants' strategies of dealing with phytopathogens. In non-angiosperm lineages, the interplay is less well understood, but current data indicate that it is intertwined to a lesser extent and the canonical JA/SA antagonism appears to be absent. Here, we used the water fern Azolla filiculoides to gain insights into the fern's JA/SA signalling and the molecular communication with its unique nitrogen fixing cyanobiont Nostoc azollae, which the fern inherits both during sexual and vegetative reproduction. By mining large-scale sequencing data, we demonstrate that Azolla has most of the genetic repertoire to produce and sense JA and SA. Using qRT-PCR on the identified biosynthesis and signalling marker genes, we show that Azolla is responsive to exogenously applied SA. Furthermore, exogenous SA application influenced the abundance and gene expression of Azolla's cyanobiont. Our data provide a framework for JA/SA signalling in ferns and suggest that SA might be involved in Azolla's communication with its vertically inherited cyanobiont.

Protists are an integral part of the Arabidopsis thaliana microbiome.

Although protists occupy a vast range of habitats and are known to interact with plants among other things via disease suppression, competition or growth stimulation, their contributions to the 'phytobiome' are not well described. To contribute to a more comprehensive picture of the plant holobiont, we examined cercozoan and oomycete taxa living in association with the model plant Arabidopsis thaliana grown in two different soils. Soil, roots, leaves and wooden toothpicks were analysed before and after surface sterilization. Cercozoa were identified using 18S rRNA gene metabarcoding, whereas the Internal Transcribed Spacer 1 was used to determine oomycetes. Subsequent analyses revealed strong spatial structuring of protist communities between compartments, although oomycetes appeared more specialized than Cercozoa. With regards to oomycetes, only members of the Peronosporales and taxa belonging to the genus Globisporangium were identified as shared members of the A. thaliana microbiome. This also applied to cercozoan taxa belonging to the Glissomonadida and Cercomonadida. We identified a strong influence by edaphic factors on the rhizosphere, but not for the phyllosphere. Distinct differences of Cercozoa found preferably in wood or fresh plant material imply specific niche adaptations. Our results highlight the importance of micro-eukaryotes for the plant holobiont.

Population Genomics in Wild Tomatoes-The Interplay of Divergence and Admixture.

Hybridization between closely related plant species is widespread, but the outcomes of hybridization are not fully understood. This study investigates phylogenetic relationships and the history of hybridization in the wild tomato clade (Solanum sect. Lycopersicon). We sequenced RNA from individuals of 38 different populations and, by combining this with published data, build a comprehensive genomic data set for the entire clade. The data indicate that many taxa are not monophyletic and many individuals are admixed due to repeated hybridization. The most polymorphic species, Solanum peruvianum, has two genetic and geographical subpopulations, while its sister species, Solanum chilense, has distinct coastal populations and reduced heterozygosity indicating a recent expansion south following speciation from S. peruvianum circa 1.25 Ma. Discontinuous populations west of 72° are currently described as S. chilense, but are genetically intermediate between S. chilense and S. peruvianum. Based upon molecular, morphological, and crossing data, we test the hypothesis that these discontinuous "S. chilense" populations are an example of recombinational speciation. Recombinational speciation is rarely reported, and we discuss the difficulties in identifying it and differentiating between alternative demographic scenarios. This discovery presents a new opportunity to understand the genomic outcomes of hybridization in plants.

How Embryophytic is the Biosynthesis of Phenylpropanoids and their Derivatives in Streptophyte Algae?

The origin of land plants from algae is a long-standing question in evolutionary biology. It is becoming increasingly clear that many characters that were once assumed to be 'embryophyte specific' can in fact be found in their closest algal relatives, the streptophyte algae. One such case is the phenylpropanoid pathway. While biochemical data indicate that streptophyte algae harbor lignin-like components, the phenylpropanoid core pathway, which serves as the backbone of lignin biosynthesis, has been proposed to have arisen at the base of the land plants. Here we revisit this hypothesis using a wealth of new sequence data from streptophyte algae. Tracing the biochemical pathway towards lignin biogenesis, we show that most of the genes required for phenylpropanoid synthesis and the precursors for lignin production were already present in streptophyte algae. Nevertheless, phylogenetic analyses and protein structure predictions of one of the key enzyme classes in lignin production, cinnamyl alcohol dehydrogenase (CAD), suggest that CADs of streptophyte algae are more similar to sinapyl alcohol dehydrogenases (SADs). This suggests that the end-products of the pathway leading to lignin biosynthesis in streptophyte algae may facilitate the production of lignin-like compounds and defense molecules. We hypothesize that streptophyte algae already possessed the genetic toolkit from which the capacity to produce lignin later evolved in vascular plants.

Signatures of selection and host-adapted gene expression of the Phytophthora infestans RNA silencing suppressor PSR2.

Phytophthora infestans is a devastating pathogen in agricultural systems. Recently, an RNA silencing suppressor (PSR2, 'Phytophthora suppressor of RNA silencing 2') has been described in P. infestans. PSR2 has been shown to increase the virulence of Phytophthora pathogens on their hosts. This gene is one of the few effectors present in many economically important Phytophthora species. In this study, we investigated: (i) the evolutionary history of PSR2 within and between species of Phytophthora; and (ii) the interaction between sequence variation, gene expression and virulence. In P. infestans, the highest PiPSR2 expression was correlated with decreased symptom expression. The highest gene expression was observed in the biotrophic phase of the pathogen, suggesting that PSR2 is important during early infection. Protein sequence conservation was negatively correlated with host range, suggesting host range as a driver of PSR2 evolution. Within species, we detected elevated amino acid variation, as observed for other effectors; however, the frequency spectrum of the mutations was inconsistent with strong balancing selection. This evolutionary pattern may be related to the conservation of the host target(s) of PSR2 and the absence of known corresponding R genes. In summary, our study indicates that PSR2 is a conserved effector that acts as a master switch to modify plant gene regulation early during infection for the pathogen's benefit. The conservation of PSR2 and its important role in virulence make it a promising target for pathogen management.

Pinpointing genes underlying annual/perennial transitions with comparative genomics.

BACKGROUND: Transitions between perennial and an annual life history occur often in plant lineages, but the genes that control whether a plant is an annual or perennial are largely unknown. To identify genes that confer differences between annuals and perennials we compared the gene content of four pairs of sister lineages (Arabidopsis thaliana/Arabidopsis lyrata, Arabis montbretiana/Arabis alpina, Arabis verna/Aubrieta parviflora and Draba nemorosa/Draba hispanica) in the Brassicaceae in which each pair contains one annual and one perennial, plus one extra annual species (Capsella rubella). RESULTS: After sorting all genes in all nine species into gene families, we identified five families in which well-annotated genes are present in the perennials A. lyrata and A. alpina, but are not present in any of the annual species. For the eleven genes in perennials in these families, an orthologous pseudogene or otherwise highly diverged gene was found in the syntenic region of the annual species in six cases. The five candidate families identified encode: a kinase, an oxidoreductase, a lactoylglutathione lyase, a F-box protein and a zinc finger protein. By comparing the active gene in the perennial to the pseudogene or heavily altered gene in the annual, dN and dS were calculated. The low dN/dS values in one kinase suggest that it became pseudogenized more recently, while the other kinase, F-box, oxidoreductase and zinc-finger became pseudogenized closer to the divergence between the annual-perennial pair. CONCLUSIONS: We identified five gene families that may be involved in the life history switch from perennial to annual. Considering the dN and dS data and whether syntenic pseudogenes were found and the potential functions of the genes, the F-box family is considered the most promising candidate for future functional studies to determine if it affects life history.