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Background Red clover (Trifolium pratense), a crop constrained by weak persistence, is the number one forage legume in Northern Europe. Whether plant loss is a sign of an increasing number of known or new pathogens, a decreasing occurrence of beneficial soil microorganisms or other factors is unclear. Here, we collected red clover root samples from Swedish conventional and organic farming sites located between latitudes 55°N and 66°N and analyzed their prokaryotic and eukaryotic communities using amplicon sequencing. We compared the organism diversity, differential abundance, hub taxa and impact of Rhizobium on fungal pathogens in relation to geographic location and farming practices. Results We found that microbial biodiversity was lowest in samples from southern Sweden and on conventional farms. Rhizobium was the most prevalent bacterial genus, followed by Sphingomonas, Mucilaginibacter, Flavobacterium and the unclassified Chloroflexi group KD4-96. The Leptodontidium, Cladosporium, Clonostachys and Tetracladium fungal genera known for endophytic, ectomycorrhizal, saprotrophic and mycoparasitic lifestyles were also frequently observed in all samples. Sixty-two potential pathogenic fungi were identified with a bias toward grass pathogens and a higher abundance in samples from conventional farms. To gain insights into the cooccurrence patterns of red clover root-associated communities, intrakingdom and interkingdom networks were constructed. Bacterial networks were dominated by known beneficial groups of bacterial species. The interaction between Rhizobium leguminosarum bv. trifolii and Aeromicrobium sp. was strongly positive in the north but negative in the south. Different co-occurrence responses were observed when fungal and bacterial data were combined, as exemplified by the interactions with Clonostachys rosea, a beneficial fungus that fluctuated in our dataset and was positively associated with species in Enterobacterales and Aeromicrobium in the north but negatively with species in the south. Conclusions We show that the microbial community is mainly shaped by geographic location and management procedures. R. leguminosarum bv. trifolii is a keystone species in samples from the northern region and Swedish organic farms. This Rhizobium species is negatively associated with all fungal pathogenic species recognized in this study. A number of previously unknown fungal species are identified, particularly those known to produce mycotoxins, which are of major concern for crop productivity and feed safety.

Phytophthora spp. cause serious damage to plants by exploiting a large number of effector proteins and small RNAs (sRNAs). Several reports have described modulation of host RNA biogenesis and defence gene expression. Here, we analysed Phytophthora infestans Argonaute (Ago) 1 associated small RNAs during potato leaf infection. Small RNAs were co-immunoprecipitated, deep sequenced and analysed against the P. infestans and potato genomes, followed by transcript analyses and transgenic assays on a predicted target. Extensive targeting of potato and pathogen-derived sRNAs to a range of mRNAs was observed, including 638 sequences coding for resistance (R) proteins in the host genome. The single miRNA encoded by P. infestans (miR8788) was found to target a potato alpha/beta hydrolase-type encoding gene (StABH1), a protein localized to the plasma membrane. Analyses of stable transgenic potato lines harbouring overexpressed StABH1 or artificial miRNA gene constructs demonstrated the importance of StABH1 during infection by P. infestans. miR8788 knock-down strains showed reduced growth on potato, and elevated StABH1 expression levels were observed when plants were inoculated with the two knock-down strains compared to the wild-type strain 88069. The findings of our study suggest that sRNA encoded by P. infestans can affect potato mRNA, thereby expanding our knowledge of the multifaceted strategies this species uses to facilitate infection.

Regulatory small RNAs (sRNAs) play important roles in many fundamental processes in plant biology such as development, fertilization and stress responses. The AGO protein family has here a central importance in gene regulation based on their capacity to associate with sRNAs followed by mRNA targeting in a sequence-complementary manner. The present study explored Argonautes (AGOs) in the Solanaceae family, with emphasis on potato, Solanum tuberosum (St). A genome-wide monitoring was performed to provide a deeper insight into gene families, genomic localization, gene structure and expression profile against the potato late blight pathogen Phytophthora infestans. Among 15 species in the Solanaceae family we found a variation from ten AGOs in Nicotiana obtusifolia to 17 in N. tabacum. Comprehensive analyses of AGO phylogeny revealed duplication of AGO1, AGO10 and AGO4 paralogs during early radiation of Solanaceae. Fourteen AGOs were identified in potato. Orthologs of AGO8 and AGO9 were missing in the potato genome. However, AGO15 earlier annotated in tomato was identified. StAGO15 differs from the other paralogs having residues of different physico-chemical properties at functionally important amino acid positions. Upon pathogen challenge StAGO15 was significantly activated and hence may play a prominent role in sRNA-based regulation of potato defense.

Degradome sequencing is commonly used to generate high-throughput information on mRNA cleavage sites mediated by small RNAs (sRNA). In our datasets of potato (Solanum tuberosum, St) and Phytophthora infestans (Pi), initial predictions generated high numbers of cleavage site predictions, which highlighted the need of improved analytic tools. Here, we present an R package based on a deep learning convolutional neural network (CNN) in a machine learning environment to optimize discrimination of false from true cleavage sites. When applying smartPARE to our datasets on potato during the infection process by the late blight pathogen, 7.3% of all cleavage windows represented true cleavages distributed on 214 sites in P. infestans and 444 sites in potato. The sRNA landscape of the two organisms is complex with uneven sRNA production and cleavage regions widespread in the two genomes. Multiple targets and several cases of complex regulatory cascades, particularly in potato, was revealed. We conclude that our new analytic approach is useful for anyone working on complex biological systems and with the interest of identifying cleavage sites particularly inferred by sRNA classes beyond miRNAs.

This short and general chapter summarizes how plants and pathogens communicate using not only proteins for recognition and signal transduction or other metabolites but also RNA molecules where small RNAs with sizes between 21 to 40 nt are most important. These small RNAs can move between plants and a range of interacting pathogenic organisms in both directions, that is, a 'cross-kingdom' communication process. The first reports on RNA-based communications between plants and plant pathogenic fungi appeared about 10 years ago. Since that time, we have learnt much about sRNA biology in plants and their function in different parasitic organisms. However, many questions on the processes involved remain unanswered. Such information is crucial in order to sustain high crop production. Besides giving a brief background, we highlight the interactions between the potato late blight pathogen and its plant host potato.

The fungal species Rhizoctonia solani belongs to the Basidiomycota division and is a ubiquitous soil-borne pathogen. It is the main agent of the damping-off disease in seedlings and causes the root and crown rot disease in sugar beets. Plant pathogens deploy small secreted proteins, called effectors, to manipulate plant immunity in order to infect the host. Here, a gene (RsCRP1) encoded a putative effector cysteine-rich protein was cloned, expressed in Cercospora beticola and used for virulence assays. The RsCRP1 gene was highly induced upon the early-infection stage of sugar beet seedlings and disease was promoted. Confocal microscopy demonstrated localization to the chloroplasts and mitochondria upon transient expression of RsCRP1 in leaves of Nicotiana benthamiana. Further, this effector was unable to induce necrosis or to suppress hypersensitive response induced by the Avr4/Cf4 complex in N. benthamiana. Overall, these data indicate that RsCRP1 is a novel effector targeting distinct plant cell organelles in order to facilitate a successful infection at the early stages of the disease development. (C) 2021 The Authors. Published by Elsevier Inc.

Sugar beets are attacked by several pathogens that cause root damages. Rhizoctonia (Greek for “root killer”) is one of them. Rhizoctonia root rot has become an increasing problem for sugar beet production and to decrease yield losses agronomical measures are adopted. Here, two partially resistant and two susceptible sugar beet genotypes were used for transcriptome analysis to discover new defense genes to this fungal disease, information to be implemented in molecular resistance breeding. Among 217 transcripts with increased expression at 2 days post-infection (dpi), three resistance-like genes were found. These genes were not significantly elevated at 5 dpi, a time point when increased expression of three Bet v I/Major latex protein (MLP) homologous genes BvMLP1, BvMLP2 and BvML3 was observed in the partially resistant genotypes. Quantitative RT-PCR analysis on diseased sugar beet seedlings validated the activity of BvMLP1 and BvMLP3 observed in the transcriptome during challenge by R. solani. The three BvMLP genes were cloned and overexpressed in Arabidopsis thaliana to further dissect their individual contribution. Transgenic plants were also compared to T-DNA mutants of orthologous MLP genes. Plants overexpressing BvMLP1 and BvMLP3 showed significantly less infection whereas additive effects were seen on Atmlp1/Atmlp3 double mutants. The data suggest that BvMLP1 and BvMLP3 may contribute to the reduction of the Rhizoctonia root rot disease in sugar beet. Impact on the defense reaction from other differential expressed genes observed in the study is discussed.

Regulatory small RNAs (sRNAs) play important roles in many fundamental processes in plant biology such as development, fertilization and stress responses. The AGO protein family has here a central importance in gene regulation based on their capacity to associate with sRNAs followed by mRNA targeting in a sequence-complementary manner. The present study explored Argonautes (AGOs) in the Solanaceae family, with emphasis on potato, Solanum tuberosum (St). A genome-wide monitoring was performed to provide a deeper insight into gene families, genomic localization, gene structure and expression profile against the potato late blight pathogen Phytophthora infestans. Among 15 species in the Solanaceae family we found a variation from ten AGOs in Nicotiana obtusifolia to 17 in N. tabacum. Comprehensive analyses of AGO phylogeny revealed duplication of AGO1, AGO10 and AGO4 paralogs during early radiation of Solanaceae. Fourteen AGOs were identified in potato. Orthologs of AGO8 and AGO9 were missing in the potato genome. However, AGO15 earlier annotated in tomato was identified. StAGO15 differs from the other paralogs having residues of different physico-chemical properties at functionally important amino acid positions. Upon pathogen challenge StAGO15 was significantly activated and hence may play a prominent role in sRNA-based regulation of potato defense.

The potato late blight pathogen Phytophthora infestans has both an asexual and a sexual mode of reproduction. In Scandinavia, the pathogen is reproducing sexually on a regular basis, whereas clonal lineages dominate in other geographical regions. This study aimed at elucidating events or key genes underlying this difference in sexual behavior. First, the transcriptomes of eight strains, known as either clonal or sexual, were compared during early stages of mating. Principal component analysis (PCA) divided the samples in two clusters A and B and a clear grouping of the mating samples together with the A1 mating type parents was observed. Induction of genes encoding DNA adenine N6-methylation (6mA) methyl-transferases clearly showed a bias toward the cluster A. In contrast, the Avrblb2 effector gene family was highly induced in most of the mating samples and was associated with cluster B in the PCA, similarly to genes coding for acetyl-transferases, which play an important role in RXLR modification prior to secretion. Avrblb2 knock-down strains displayed a reduction in virulence and oospore formation, suggesting a role during the mating process. In conclusion, a number of gene candidates important for the reproductive processes were revealed. The results suggest a possible epigenetic influence and involvement of specific RXLR effectors in mating-related processes.

Plasmodiophora brassicae is a soil-borne pathogen that belongs to Rhizaria, an almost unexplored eukaryotic organism group. This pathogen requires a living host for growth and multiplication, which makes molecular analysis further complicated. To broaden our understanding of a plasmodiophorid such as P. brassicae, we here chose to study immunophilins, a group of proteins known to have various cellular functions, including involvement in plant defense and pathogen virulence. Searches in the P. brassicae genome resulted in 20 putative immunophilins comprising of 11 cyclophilins (CYPs), 7 FK506-binding proteins (FKBPs) and 2 parvulin-like proteins. RNAseq data showed that immunophilins were differentially regulated in enriched life stages such as germinating spores, maturing spores, and plasmodia, and infected Brassica hosts (B. rapa, B. napus and B. oleracea). PbCYP3 was highly induced in all studied life stages and during infection of all three Brassica hosts, and hence was selected for further analysis. PbCYP3 was heterologously expressed in Magnaporthe oryzae gene-inactivated ΔCyp1 strain. The new strain ΔCyp1+ overexpressing PbCYP3 showed increased virulence on rice compared to the ΔCyp1 strain. These results suggest that the predicted immunophilins and particularly PbCYP3 are activated during plant infection. M. oryzae is a well-studied fungal pathogen and could be a valuable tool for future functional studies of P. brassicae genes, particularly elucidating their role during various infection phases. Electronic supplementary material The online version of this article (10.1007/s00438-017-1395-0) contains supplementary material, which is available to authorized users.

SummaryPhytophthoraspp. incite serious plant damages by exploiting a large number of effector proteins and small RNAs (sRNAs). Several reports are describing modulation of host RNA biogenesis and defence gene expression. Here, we analysedP. infestansArgonaute (Ago) 1 associated small RNAs during potato leaf infection.sRNAs were co-immunoprecipitated, deep sequenced and analysed against theP. infestansand potato genomes, followed by transgenic and biochemical analyses on a predicted host target.Extensive targeting of potato and pathogen-derived sRNAs to a large number of mRNAs was observed, including 206 sequences coding for resistance (R) proteins in the host genome. The single miRNA encoded byP. infestans(miR8788) was found to target a potato lipase-like membrane protein-encoding gene (StLL1) localized to the tonoplast. Analyses of stable transgenic potato lines harbouring overexpressedStLL1or artificial miRNA gene constructs demonstrated the importance of StLL1 during infection byP. infestans. Similarly, a miR8788 knock-down strain showed reduced growth on potato compared to the wild-type strain 88069.The data suggest that sRNA encoded byP. infestanscan affect potato mRNA and thereby promote disease. Knowledge of the impact of pathogen small RNAs in plant defence mechanisms is of major significance to succeed in improved disease control management.

Rhizoctonia solani (Rs) is a soil-borne pathogen with a broad host range. This pathogen incites a wide range of disease symptoms. Knowledge regarding its infection process is fragmented, a typical feature for basidiomycetes. In this study, we aimed at identifying potential fungal effectors and their function. From a group of 11 predicted single gene effectors, a rare lipoprotein A (RsRlpA), from a strain attacking sugar beet was analyzed. The RsRlpA gene was highly induced upon early-stage infection of sugar beet seedlings, and heterologous expression in Cercospora beticola demonstrated involvement in virulence. It was also able to suppress the hypersensitive response (HR) induced by the Avr4/Cf4 complex in transgenic Nicotiana benthamiana plants and functioned as an active protease inhibitor able to suppress Reactive Oxygen Species (ROS) burst. This effector contains a double-psi beta-barrel (DPBB) fold domain, and a conserved serine at position 120 in the DPBB fold domain was found to be crucial for HR suppression. Overall, R. solani seems to be capable of inducing an initial biotrophic stage upon infection, suppressing basal immune responses, followed by a switch to necrotrophic growth. However, regulatory mechanisms between the different lifestyles are still unknown.

Drought stress often leads to reduced yields and is a perilous delimiter for expanded cultivation and increased productivity of sweet potato. Cell wall stabilization proteins have been identified to play a pivotal role in mechanical stabilization during desiccation stress mitigation in plants. They are involved in numerous cellular processes that modify cell wall properties to tolerate the mechanical stress during dehydration. This provides a plausible approach to engineer crops for enhanced stable yields under adverse climatic conditions. In this study, we genetically engineered sweet potato cv. Jewel with XvSap1 gene encoding a protein related to cell wall stabilization, isolated from the resurrection plant Xerophyta viscosa, under stress-inducible XvPSap1 promoter via Agrobacterium-mediated transformation. Detection of the transgene by PCR, Southern blot, and quantitative real-time PCR (qRT-PCR) analyses revealed the integration of XvSap1 in the three independent events. Phenotypic evaluation of shoot length, number of leaves, and yield revealed that the transgenic plants grew better than the wild-type plants under drought stress. Assessment of biochemical indices during drought stress showed higher levels of chlorophyll, free proline, and relative water content and decreased lipid peroxidation in transgenic plants than in wild types. Our findings demonstrate that XvSap1 enhances drought tolerance in transgenic sweet potato without causing deleterious phenotypic and yield changes. The transgenic drought-tolerant sweet potato lines provide a valuable resource as a drought-tolerant crop on arid lands of the world.

Key message Drought stress in sweetpotato could be overcome by introducing XvSap1 gene from Xerophyta viscosa.Drought stress often leads to reduced yields and is perilous delimiter for expanded cultivation and increased productivity of sweetpotato. Cell wall stabilization proteins have been identified to play a pivotal role in mechanical stabilization during desiccation stress mitigation. They are involved in myriad cellular processes that modify the cell wall properties to tolerate the mechanical stress during dehydration in plants. This provides a possible approach to engineer crops for enhanced stable yields under adverse climatic conditions. In this study, we introduced the XvSap1 gene isolated from Xerophyta viscosa, a resurrection plant into sweetpotato by Agrobacterium-mediated transformation. Detection of the transgene by PCR coupled with Southern blot revealed the integration of XvSap1 in the three independent events. Sweetpotato plants expressing the XvSap1 gene exhibited superior growth performance such as shoot length, number of leaves and yield than the wild type plants under drought stress. Quantitative real time-PCR results confirmed higher expression of the XvSap1 gene in XSP1 transgenic plants imposed with drought stress. In addition, the transgenic plants had increased levels of chlorophyll, free proline and relative water content but malonaldehyde content was decreased under drought stress compared to wild type plants. Conjointly, our findings show that XvSap1 can enhance drought resilience without causing deleterious phenotypic and yield changes, thus providing a promising candidate target for improving the drought tolerance of sweetpotato cultivars through genetic engineering. The transgenic drought tolerant sweetpotato line provides a valuable resource as drought tolerant crop on arid lands of the world.

Extensive efforts are being made to develop oilseed rape cultivars with resistance to Verticillium wilt. Evaluation of these cultivars involves either dipping roots of young plants in a conidial suspension followed by replanting in soil, or sowing the seed directly in artificially infested soil. These methods for screening for resistance take a long time and are expensive. The objective of this research was to develop a rapid and reliable method of screening cultivars for resistance to Verticillium wilt. A box test method in which 4-day-old seedlings were inoculated and grown under sterile conditions in double magenta boxes was developed. The Brassica napus cultivars Accord, Express, Hanna, and Libraska were inoculated with isolates of Verticillium longisporum or V. dahliae. The results from the box test method were compared with root-dip inoculation of seedlings grown in a soil mixture under greenhouse conditions. In addition, plants from the box test were randomly selected and transferred to pots containing sterile soil to permit comparison of plants from the box test at similar developmental stage with plants in the root-dip method. Disease evaluation was based on the number of days after transferring seedlings into the boxes that root browning, root and shoot blackening, and wilting of cotyledons developed. Additional disease evaluation was based on a 0 to 5 disease severity scale based on the percentage of wilted leaves (number) and/or the percentage of stunting (mm) of inoculated plants compared with noninoculated plants. Disease severity was higher on Hanna than on Express, and isolates of V. longisporum were more virulent than isolates of V. dahliae. There was a significant correlation (r = 0.93) of disease rating between the traditional root-dip method and the new box test method. Analysis of variance revealed that the interaction between cultivars and isolates was highly significant (P ≤ 0.01), whereas method of inoculation did not affect the results. In the box test, plants were rated after 25 days, which is at least 3.5 times more rapid than the root-dip method.

Rhizoctonia solani is a fungal species that belongs to the fungal division Basidiomycota. It is a soil-borne pathogen that attacks a broad range of plant species and crops. Disease symptoms are commonly seen as damping off of seedlings and root rot, although it can infect plants at any developmental stage. Despite the severity of this disease, many aspects in R. solani infection biology remain unclear. Here we investigated the role of a LysM effector, previously predicted from the genome of a R. solani AG2-2IIIB strain that has sugar beet as a host. Gene expression analysis showed that RsLysM was highly induced upon sugar beet infection. When RsLysM was heterologously expressed in Cercospora beticola, necrotic lesion size and fungal colonization ability were increased, indicating a role in virulence. RsLysM displayed chitin-binding affinity and suppression of chitin-triggered immunity but could not protect hyphae from hydrolysis. Overall, this study is the first characterization of a LysM effector from Basidiomycota, suggesting that this necrotrophic fungal species relies on perturbation of chitin-triggered immunity to establish a successful infection.

Phytophthora spp. encode large sets of effector proteins and distinct populations of small RNAs (sRNAs). Recent evidence has suggested that pathogen-derived sRNAs can modulate the expression of plant defense genes. Here, we studied the sRNA classes and functions associated with Phytophthora infestans Argonaute (Ago) proteins. sRNAs were co-immunoprecipitated with three PiAgo proteins and deep sequenced. Twenty- to twenty-two-nucleotide (nt) sRNAs were identified as the main interaction partners of PiAgo1 and high enrichment of 24-26-nt sRNAs was seen in the PiAgo4-bound sample. The frequencies and sizes of transposable element (TE)-derived sRNAs in the different PiAgo libraries suggested diversified roles of the PiAgo proteins in the control of different TE classes. We further provide evidence for the involvement of PiAgo1 in the P. infestans microRNA (miRNA) pathway. Protein-coding genes are probably regulated by the shared action of PiAgo1 and PiAgo5, as demonstrated by analysis of differential expression. An abundance of sRNAs from genes encoding host cell death-inducing Crinkler (CRN) effectors was bound to PiAgo1, implicating this protein in the regulation of the expanded CRN gene family. The data suggest that PiAgo1 plays an essential role in gene regulation and that at least two RNA silencing pathways regulate TEs in the plant-pathogenic oomycete P. infestans.

Rhizoctonia (in Greek root-killer) species and particularly R. solani attacks a broad range of plant species and crops. It belongs to Basidiomycota and is a soil-borne pathogen causing mainly damping-off in seedlings, although it can infect plants in any stage. It does not form any kind of asexual spores while the sexual form (Thanatephorus cucumeris) is extremely rare. However, it forms microsclerotia, able to survive in soil and plant debris for a long period and under harsh environmental conditions. Despite the severity of this disease, many aspects in R. solani infection biology still remain to be elucidated. In this study, we investigated the role of two effector candidates, derived from a R. solani AG2-2IIIB strain isolated from infected sugar beet plants. Gene expression analysis showed that genes encoding for a LysM effector and a RlpA-like protein were induced upon early infection stages, while their heterologous expression in Cercospora beticola contributed to virulence. Furthermore, the RsLysM effector showed chitin binding affinity, suppressing chitin-triggered immunity but could not protect hyphae from hydrolysis. The RlpA-like effector is homologous to a papain-like inhibitor and to a barwin-like endoglucanase, while our data showed that it was able to suppress hypersensitive response in Nicotiana bethamiana leaves. Overall, this study provides us with valuable information on R. solani infection biology towards understanding the molecular mechanisms in this pathosystem.

Sweetpotato is a significant crop which is widely cultivated particularly in the developing countries with high and stable yield. However, drought stress is a major limiting factor that antagonistically influences the crop’s productivity. Dehydration stress caused by drought causes aggregation of reactive oxygen species (ROS) in plants, and aldose reductases are first-line safeguards against ROS caused by oxidative stress. In the present study, we generated transgenic sweetpotato plants expressing aldose reductase, XvAld1 isolated from Xerophyta viscosa under the control of a stress-inducible promoter via Agrobacterium-mediated transformation. Our results demonstrated that the transgenic sweetpotato lines displayed significant enhanced tolerance to simulated drought stress and enhanced recuperation after rehydration contrasted with wild-type plants. In addition, the transgenic plants exhibited improved photosynthetic efficiency, higher water content and more proline accumulation under dehydration stress conditions compared with wild-type plants. These results demonstrate that exploiting the XvAld1 gene is not only a compelling and attainable way to improve sweetpotato tolerance to drought stresses without causing any phenotypic imperfections but also a promising gene candidate for more extensive crop improvement.

Plasmodiophora brassicae causes clubroot on cruciferous plants and causes worldwide huge economical losses on important Brassica crops. P. brassicae infection produces large root galls, the clubroots, which can also affect the upper plant parts by reduced water and nutrient uptake and redirection of assimilates from leaves to roots. P. brassicae is an obligate biotrophic protist in the plasmodiophorids within the eukaryote supergroup of Rhizaria and is unrelated to other known plant pathogens. Plasmodiophorids can be parasites of plants and oomycetes. The recently released genome of P. brassicae is only the third in the poorly studied Rhizaria and the first plant pathogenic genome of this eukaryotic group. The P. brassicae genome was estimated to be 25.5 Mb in size and predicted to contain 9730 gene models. A transcriptome of P. brassicae and Spongospora subterranea, the potato scab pathogen was also presented. Consequently, for the first time large scale data for a eukaryotic plant pathogen group outside the fungi and oomycetes are now available. This review highlights selected characteristics of the P. brassicae genome including molecular events shown or predicted to take place in each phase of its life-cycle, such as manipulation of: 1) host primary metabolism, 2) plant hormone homeostasis, and 3) plant defense. Further, future directions and challenges in the P. brassicae and plasmodiophorid genomic research are discussed.

International audience; The SAR group (Stramenopila, Alveolata, Rhizaria) is one of the largest clades in the tree of eukaryotes and includes a great number of parasitic lineages. Rhizarian parasites are obligate and have devastating effects on commercially important plants and animals but despite this fact, our knowledge of their biology and evolution is limited. Here, we present rhizarian transcriptomes from all major parasitic lineages in order to elucidate their evolutionary relationships using a phylogenomic approach. Our results suggest that Ascetosporea, parasites of marine invertebrates, are sister to the novel clade Apofilosa. The phytomyxean plant parasites branch sister to the vampyrellid algal ectoparasites in the novel clade Phytorhiza. They also show that Ascetosporea + Apofilosa + Retaria + Filosa + Phytorhiza form a monophyletic clade, although the branching pattern within this clade is difficult to resolve and appears to be model-dependent. Our study does not support the monophyly of the rhizarian parasitic lineages (Endomyxa), suggesting independent origins for rhizarian animal and plant parasites.

Highlight A host-induced gene-silencing strategy for controlling potato late blight is presented, a plant disease that conventionally requires regular application of fungicides at high rates., Phytophthora infestans is an oomycete that causes severe damage to potato, and is well known for its ability to evolve rapidly in order to overcome resistant potato varieties. An RNA silencing strategy was evaluated here to clarify if small interfering RNA homologous to selected genes in P. infestans could be targeted from the plant host to reduce the magnitude of the infection. As a proof-of-concept, a hairpin RNA (hp-RNA) construct using the GFP marker gene was designed and introduced in potato. At 72 hpi, a 55-fold reduction of the signal intensity of a corresponding GFP expressing P. infestans strain on leaf samples of transgenic plants, compared with wild-type potato, was detected. This suggests that an RNA interference construct in the potato host could be processed and target a transcript of the pathogen. Three genes important in the infection process of P. infestans, PiGPB1, PiCESA2, and PiPEC, together with PiGAPDH taking part in basic cell maintenance were subsequently tested using an analogous transgenic strategy. Out of these gene candidates, the hp-PiGPB1 targeting the G protein β-subunit (PiGPB1) important for pathogenicity resulted in most restricted disease progress. Further, Illumina sequencing of inoculated transgenic potato leaves revealed sRNAs of 24/25 nt size homologous to the PiGPB1 gene in the transgenic plants indicating post-transcriptional silencing of the target gene. The work demonstrates that a host-induced gene-silencing approach is functional against P. infestans but is highly dependent on target gene for a successful outcome. This finding broadens the arsenal of control strategies to this important plant disease.

Background Brassica plant species are attacked by a number of pathogens; among them, the ones with a soil-borne lifestyle have become increasingly important. Verticillium stem stripe caused by Verticillium longisporum is one example. This fungal species is thought to be of a hybrid origin, having a genome composed of combinations of lineages denominated A and D. In this study we report the draft genomes of 2 V. longisporum field isolates sequenced using the Illumina technology. Genomic characterization and lineage composition, followed by selected gene analysis to facilitate the comprehension of its genomic features and potential effector categories were performed. Results The draft genomes of 2 Verticillium longisporum single spore isolates (VL1 and VL2) have an estimated ungapped size of about 70 Mb. The total number of protein encoding genes identified in VL1 was 20,793, whereas 21,072 gene models were predicted in VL2. The predicted genome size, gene contents, including the gene families coding for carbohydrate active enzymes were almost double the numbers found in V. dahliae and V. albo-atrum. Single nucleotide polymorphisms (SNPs) were frequently distributed in the two genomes but the distribution of heterozygosity and depth was not independent. Further analysis of potential parental lineages suggests that the V. longisporum genome is composed of two parts, A1 and D1, where A1 is more ancient than the parental lineage genome D1, the latter being more closer related to V. dahliae. Presence of the mating-type genes MAT1–1-1 and MAT1–2-1 in the V. longisporum genomes were confirmed. However, the MAT genes in V. dahliae, V. albo-atrum and V. longisporum have experienced extensive nucleotide changes at least partly explaining the present asexual nature of these fungal species. Conclusions The established draft genome of V. longisporum is comparatively large compared to other studied ascomycete fungi. Consequently, high numbers of genes were predicted in the two V. longisporum genomes, among them many secreted proteins and carbohydrate active enzyme (CAZy) encoding genes. The genome is composed of two parts, where one lineage is more ancient than the part being more closely related to V. dahliae. Dissimilar mating-type sequences were identified indicating possible ancient hybridization events. Electronic supplementary material The online version of this article (doi: 10.1186/s12864-017-4407-x) contains supplementary material, which is available to authorized users.

Verticillium species are soilborne plant pathogens, responsible for big yield losses worldwide. Here, we report improved procedures to generate DNA from Verticillium species imbedded in farm soils. Using new genomic sequence information, primers for V. dahliae, V. albo-atrum, V. tricorpus, and V. longisporum were designed. In a survey of 429 samples from intensively farmed soil of two Swedish regions, only V. dahliae and V. longisporum were identified. A bias towards V. longisporum (40%) was seen in the south, whereas V. dahliae was more frequent in the western region (19%). Analyses of soil and leaf samples from 20 sugar beet fields, where foliar wilting had been observed, revealed V. dahliae DNA in all leaf and soil samples and V. longisporum in 18 soil samples, illustrating host choice and longevity of the V. longisporum microsclerotia. This study demonstrates the applicability of new molecular diagnostic tools that are important for growers of variable crops.

Clubroot disease caused by Plasmodiophora brassicae is one of the most important diseases of cultivated brassicas. P. brassicae occurs in pathotypes which differ in the aggressiveness towards their Brassica host plants. To date no DNA based method to distinguish these pathotypes has been described. In 2011 polymorphism within the 28S rDNA of P. brassicae was reported which potentially could allow to distinguish pathotypes without the need of time-consuming bioassays. However, isolates of P. brassicae from around the world analysed in this study do not show polymorphism in their LSU rDNA sequences. The previously described polymorphism most likely derived from soil inhabiting Cercozoa more specifically Neoheteromita-like glissomonads. Here we correct the LSU rDNA sequence of P. brassicae. By using FISH we demonstrate that our newly generated sequence belongs to the causal agent of clubroot disease.

Opponents of genetically modified crops continue to raise concerns about risk, despite 20 years of research disproving their claims. Science should close the book on risk research and turn to studying the economic and environmental benefits of agricultural biotechnology

In order to understand the underlaying causes of new severe turcicum leaf blight outbreaks in East Africa, a survey was undertaken in Uganda to examine the sorghum—Setosphaeria turcica interaction in terms of disease severity and incidence, the overall fungal population structure, and new resistant resources. Highest disease severities were recorded on caudatum accessions, whereas kafir genotypes were most resistant. The disease was more severe in the most humid farmlands compared to moderately dry agro-ecologies. In districts with wide adoption of the Epuripur variety a very high incidence (100%) of turcicum leaf blight was found. The two S. turcica mating type genes MAT1-1 and MAT1-2 assessed on fungal isolates deriving from both sorghum and maize diseased leaves were found in 20 of 23 districts sampled and in equal proportions. Upon cross inoculation on maize differential lines, four S. turcica isolates were identified as race 1, two as race 2, and one isolate corresponded to race 0 and race 3, respectively. The remaining 10 S. turcica isolates did not cause any disease symptoms on the maize lines assessed. Highly resistant accessions originating from a regional collection were found among the five sorghum races (kafir, guinea, caudatum, bicolor and durra), and are now implemented in new sorghum disease resistance programs.

Gene silencing may have a direct or indirect impact on many biological processes in eukaryotic cells, and is a useful tool for the determination of the roles of specific genes. In this article, we report silencing in Phytophthora infestans, an oomycete pathogen of potato and tomato. Gene silencing is known to occur in P. infestans, but its genetic basis has yet to be determined. Genes encoding the major components of the RNA interference (RNAi) pathway, Dicer-like (Pidcl1), Argonaute (Piago1-5) and RNA-directed RNA polymerase (Pirdr1), were identified in the P. infestans genome by comparative genomics, together with families of other genes potentially involved in gene silencing, such as histone deacetylases, histone methyltransferases, DEAD helicases, chromodomain proteins and a class 1 RNaseIII. Real-time reverse transcription-polymerase chain reaction demonstrated transcript accumulation for all candidate genes throughout the asexual lifecycle and plant infection, but at different levels of mRNA abundance. A functional assay was developed in which silencing of the sporulation-associated Picdc14 gene was released by the treatment of protoplasts with in vitro-synthesized double-stranded RNAs homologous to Pidcl1, Piago1/2 and histone deacetylase Pihda1. These results suggest that the components of gene silencing, namely Dicer-like, Argonaute and histone deacetylase, are functional in P. infestans. Our data demonstrate that this oomycete possesses canonical gene silencing pathways similar to those of other eukaryotes.

Fungal and oomycete plant pathogens translocate effector proteins into host cells to establish infection. However, virulence targets and modes of action of their effectors are unknown. Effector AVR3a from potato blight pathogen Phytophthora infestans is translocated into host cells and occurs in two forms: AVR3a KI , which is detected by potato resistance protein R3a, strongly suppresses infestin 1 (INF1)-triggered cell death (ICD), whereas AVR3a EM , which evades recognition by R3a, weakly suppresses host ICD. Here we show that AVR3a interacts with and stabilizes host U-box E3 ligase CMPG1, which is required for ICD. In contrast, AVR3a KI/Y147del , a mutant with a deleted C-terminal tyrosine residue that fails to suppress ICD, cannot interact with or stabilize CMPG1. CMPG1 is stabilized by the inhibitors MG132 and epoxomicin, indicating that it is degraded by the 26S proteasome. CMPG1 is degraded during ICD. However, it is stabilized by mutations in the U-box that prevent its E3 ligase activity. In stabilizing CMPG1, AVR3a thus modifies its normal activity. Remarkably, given the potential for hundreds of effector genes in the P. infestans genome, silencing Avr3a compromises P. infestans pathogenicity, suggesting that AVR3a is essential for virulence. Interestingly, Avr3a silencing can be complemented by in planta expression of Avr3a KI or Avr3a EM but not the Avr3a KI/Y147del mutant. Our data provide genetic evidence that AVR3a is an essential virulence factor that targets and stabilizes the plant E3 ligase CMPG1, potentially to prevent host cell death during the biotrophic phase of infection.

A collection of Mycosphaerella fijiensis, in total 71 isolates from 11 countries, of which 54 isolates were derived from the Ugandan Lake Victoria region, were analysed with regard to molecular markers and benomyl resistance to characterize the population structure. Forty-seven alleles from seven restriction fragment length polymorphism (PCR-RFLP) and five microsatellite markers were scored and utilized in the study. Using analysis of molecular variance (AMOVA), no significant genetic differentiation could be found between isolates from different leaves compared to isolates from a single leaf (P = 0.07). Exact tests of the allelic frequencies revealed no differences between isolates from different districts or between single-leaf-derived isolates compared to isolates derived from different leaves (P > 0.05 in all cases except two, which, however, were shown to be of type-I error). Resistance to benomyl, analysed both by PCR and fungicide screening, revealed that all isolates were susceptible. Compiled information from the various datasets implied that the Ugandan M. fijiensis population in the Lake Victoria Basin constitutes a homogenous population, probably as the result of a recent founder event.

Spot blotch, caused by Bipolaris sorokiniana, has become an increasingly important disease on barley in Sweden. In order to enhance our knowledge of this disease, eight Swedish B. sorokiniana isolates were assessed for their virulence pathotype on 30 barley genotypes including eight lines of mutant origin, designated B. sorokiniana tolerant (bst). A leaf screen assay revealed the presence of virulence groups 1 and 7 in Sweden. Several mutant genotypes demonstrated enhanced resistance responses (P = 0.0001), of which bstl:3 had the lowest leaf scoring value to the most virulent isolate, Swe2 (virulence group 7). To evaluate the host root responses, two contrasting isolates Swe2 and Swe4 (virulence group 7 and 1) were used to inoculate a set of eight differential lines, including four promising mutant lines from the leaf screen. Mutant bstl:6 was significantly less susceptible (P = 0.001) compared with the other barley genotypes tested, independent of the two isolates used. Mutants bst1, bst1:3 and bstl:4, which displayed less or no spots, are also considered promising genotypes for further breeding attempts.

Here, we describe the rapid cloning of a plant gene, Leptosphaeria maculans 3 (RLM3(Col)), which encodes a putative Toll interleukin-1 receptor-nucleotide binding (TIR-NB) class protein, which is involved in defence against the fungal pathogen L. maculans and against three other necrotrophic fungi. We have, through microarray-based case control bulk segregant comparisons of transcriptomes in pools of Col-0 x An-1 progeny, identified the absence of a locus that causes susceptibility in An-1. The significance of this locus on chromosome 4 for L. maculans resistance was supported by PCR-based mapping, and denoted resistance to RLM3(Col). Differential susceptible phenotypes in four independent T-DNA insertion lines support the hypothesis that At4g16990 is required for RLM3(Col) function. The mutants in RLM3(Col) also exhibited an enhanced susceptibility to Botrytis cinerea, Alternaria brassicicola and Alternaria brassicae. Complementations of An-1 and T-DNA mutants using overexpression of a short transcript lacking the NB-ARC domain, or a genomic clone, restored resistance to all necrotrophic fungi. The elevated expression of RLM3(Col) on B. cinerea-susceptible mutants further suggested convergence in signalling and gene regulation between defence against B. cinerea and L. maculans. In the case of L. maculans, RLM3(Col) is required for efficient callose deposition downstream of RLM1(Col).