Thermoprotection by a cell membrane-localized metacaspase in a green alga.

Caspases are restricted to animals, while other organisms, including plants, possess metacaspases (MCAs), a more ancient and broader class of structurally related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis (Arabidopsis thaliana) with 9 MCAs with partially redundant activities. In contrast to streptophytes, most chlorophytes contain only 1 or 2 uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigated CrMCA-II, the single type-II MCA from the model chlorophyte Chlamydomonas (Chlamydomonas reinhardtii). Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlated with its dimerization. Most of CrMCA-II in the cell was present as a proenzyme (zymogen) attached to the plasma membrane (PM). Deletion of CrMCA-II by genome editing compromised thermotolerance, leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restored thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we connected the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

Phytophthora infestans Ago1-associated miRNA promotes potato late blight disease.

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.

Phytophthora infestans Argonaute 1 binds microRNA and small RNAs from effector genes and transposable elements.

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.

Plant-mediated gene silencing restricts growth of the potato late blight pathogen Phytophthora infestans.

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 ß-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.

Fragmentation of tRNA in Phytophthora infestans asexual life cycle stages and during host plant infection.

BACKGROUND: The oomycete Phytophthora infestans possesses active RNA silencing pathways, which presumably enable this plant pathogen to control the large numbers of transposable elements present in its 240 Mb genome. Small RNAs (sRNAs), central molecules in RNA silencing, are known to also play key roles in this organism, notably in regulation of critical effector genes needed for infection of its potato host. RESULTS: To identify additional classes of sRNAs in oomycetes, we mapped deep sequencing reads to transfer RNAs (tRNAs) thereby revealing the presence of 19-40 nt tRNA-derived RNA fragments (tRFs). Northern blot analysis identified abundant tRFs corresponding to half tRNA molecules. Some tRFs accumulated differentially during infection, as seen by examining sRNAs sequenced from P. infestans-potato interaction libraries. The putative connection between tRF biogenesis and the canonical RNA silencing pathways was investigated by employing hairpin RNA-mediated RNAi to silence the genes encoding P. infestans Argonaute (PiAgo) and Dicer (PiDcl) endoribonucleases. By sRNA sequencing we show that tRF accumulation is PiDcl1-independent, while Northern hybridizations detected reduced levels of specific tRNA-derived species in the PiAgo1 knockdown line. CONCLUSIONS: Our findings extend the sRNA diversity in oomycetes to include fragments derived from non-protein-coding RNA transcripts and identify tRFs with elevated levels during infection of potato by P. infestans.

Dominance of Mating Type A1 and Indication of Epigenetic Effects During Early Stages of Mating in Phytophthora infestans.

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.

Nucleomorph Small RNAs in Cryptophyte and Chlorarachniophyte Algae.

The regulation of gene expression and RNA maturation underlies fundamental processes such as cell homeostasis, development, and stress acclimation. The biogenesis and modification of RNA is tightly controlled by an array of regulatory RNAs and nucleic acid-binding proteins. While the role of small RNAs (sRNAs) in gene expression has been studied in-depth in select model organisms, little is known about sRNA biology across the eukaryotic tree of life. We used deep sequencing to explore the repertoires of sRNAs encoded by the miniaturized, endosymbiotically derived "nucleomorph" genomes of two single-celled algae, the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. A total of 32.3 and 35.3 million reads were generated from G. theta and B. natans, respectively. In G. theta, we identified nucleomorph U1, U2, and U4 spliceosomal small nuclear RNAs (snRNAs) as well as 11 C/D box small nucleolar RNAs (snoRNAs), five of which have potential plant and animal homologs. The snoRNAs are predicted to perform 2'-O methylation of rRNA (but not snRNA). In B. natans, we found the previously undetected 5S rRNA as well as six orphan sRNAs. Analysis of chlorarachniophyte snRNAs shed light on the removal of the miniature 18-21 nt introns found in B. natans nucleomorph genes. Neither of the nucleomorph genomes appears to encode RNA pseudouridylation machinery, and U5 snRNA cannot be found in the cryptophyte G. theta. Considering the central roles of U5 snRNA and RNA modifications in other organisms, cytoplasm-to-nucleomorph RNA shuttling in cryptophyte algae is a distinct possibility.

Phenotypic diversification by gene silencing in Phytophthora plant pathogens.

Advances in genome sequencing technologies have enabled generation of unprecedented information on genome content and organization. Eukaryote genomes in particular may contain large populations of transposable elements (TEs) and other repeated sequences. Active TEs can result in insertional mutations, altered transcription levels and ectopic recombination of DNA. The genome of the oomycete plant pathogen, Phytophthora infestans, contains vast numbers of TE sequences. There are also hundreds of predicted disease-promoting effector proteins, predominantly located in TE-rich genomic regions. Expansion of effector gene families is also a genomic signature of related oomycetes such as P. sojae. Deep sequencing of small RNAs (sRNAs) from P. infestans has identified sRNAs derived from all families of transposons, highlighting the importance of RNA silencing for maintaining these genomic invaders in an inactive form. Small RNAs were also identified from specific effector encoding genes, possibly leading to RNA silencing of these genes and variation in pathogenicity and virulence toward plant resistance genes. Similar findings have also recently been made for the distantly related species, P. sojae. Small RNA "hotspots" originating from arrays of amplified gene sequences, or from genes displaying overlapping antisense transcription, were also identified in P. infestans. These findings suggest a major role for RNA silencing processes in the adaptability and diversification of these economically important plant pathogens. Here we review the latest progress and understanding of gene silencing in oomycetes with emphasis on transposable elements and sRNA-associated events.

Evidence for small RNAs homologous to effector-encoding genes and transposable elements in the oomycete Phytophthora infestans.

Phytophthora infestans is the oomycete pathogen responsible for the devastating late blight disease on potato and tomato. There is presently an intense research focus on the role(s) of effectors in promoting late blight disease development. However, little is known about how they are regulated, or how diversity in their expression may be generated among different isolates. Here we present data from investigation of RNA silencing processes, characterized by non-coding small RNA molecules (sRNA) of 19-40 nt. From deep sequencing of sRNAs we have identified sRNAs matching numerous RxLR and Crinkler (CRN) effector protein genes in two isolates differing in pathogenicity. Effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Small RNAs in P. infestans grouped into three clear size classes of 21, 25/26 and 32 nt. Small RNAs from all size classes mapped to RxLR effector genes, but notably 21 nt sRNAs were the predominant size class mapping to CRN effector genes. Some effector genes, such as PiAvr3a, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. The P. infestans genome is rich in transposable elements, and the majority of sRNAs of all size classes mapped to these sequences, predominantly to long terminal repeat (LTR) retrotransposons. RNA silencing of Dicer and Argonaute genes provided evidence that generation of 21 nt sRNAs is Dicer-dependent, while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, we identified six microRNA (miRNA) candidates from our sequencing data, their precursor sequences from the genome sequence, and target mRNAs. These miRNA candidates have features characteristic of both plant and metazoan miRNAs.