Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq).

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi-amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi-amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen-enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single-molecule real-time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi-amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full-length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi-amr1.

Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl-CoA binding protein.

Microalgal oils in the form of triacylglycerols (TAGs) are broadly used as nutritional supplements and biofuels. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of acyl-CoA-dependent biosynthesis of TAG, and is considered a key target for manipulating oil production. Although a growing number of DGAT1s have been identified and over-expressed in some algal species, the detailed structure-function relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structure-function features of the hydrophilic N-terminal domain of DGAT1 from the green microalga Chromochloris zofingiensis (CzDGAT1). The results indicated that the N-terminal domain of CzDGAT1 was less disordered than those of the higher eukaryotic enzymes and its partial truncation or complete removal could substantially decrease enzyme activity, suggesting its possible role in maintaining enzyme performance. Although the N-terminal domains of animal and plant DGAT1s were previously found to bind acyl-CoAs, replacement of CzDGAT1 N-terminus by an acyl-CoA binding protein (ACBP) could not restore enzyme activity. Interestingly, the fusion of ACBP to the N-terminus of the full-length CzDGAT1 could enhance the enzyme affinity for acyl-CoAs and augment protein accumulation levels, which ultimately drove oil accumulation in yeast cells and tobacco leaves to higher levels than the full-length CzDGAT1. Overall, our findings unravel the distinct features of the N-terminus of algal DGAT1 and provide a strategy to engineer enhanced performance in DGAT1 via protein fusion, which may open a vista in generating improved membrane-bound acyl-CoA-dependent enzymes and boosting oil biosynthesis in plants and oleaginous microorganisms.

Chlamydomonas cell cycle mutant crcdc5 over-accumulates starch and oil.

The use of algal biomass for biofuel production requires improvements in both biomass productivity and its energy density. Green microalgae store starch and oil as two major forms of carbon reserves. Current strategies to increase the amount of carbon reserves often compromise algal growth. To better understand the cellular mechanisms connecting cell division to carbon storage, we examined starch and oil accumulation in two Chlamydomonas mutants deficient in a gene encoding a homolog of the Arabidopsis Cell Division Cycle 5 (CDC5), a MYB DNA binding protein known to be involved in cell cycle in higher plants. The two crcdc5 mutants (crcdc5-1 and crcdc5-2) were found to accumulate significantly higher amount of starch and oil than their corresponding parental lines. Flow cytometry analysis on synchronized cultures cultivated in a diurnal light/dark cycle revealed an abnormal division of the two mutants, characterized by a prolonged S/M phase, therefore demonstrating its implication in cell cycle in Chlamydomonas. Taken together, these results suggest that the energy saved by a slowdown in cell division is used for the synthesis of reserve compounds. This work highlights the importance in understanding the interplay between cell cycle and starch/oil homeostasis, which should have a critical impact on improving lipid/starch productivity.

Sequencing and comparative analysis of three Chlorella genomes provide insights into strain-specific adaptation to wastewater.

Microalgal Chlorella has been demonstrated to process wastewater efficiently from piggery industry, yet optimization through genetic engineering of such a bio-treatment is currently challenging, largely due to the limited data and knowledge in genomics. In this study, we first investigated the differential growth rates among three wastewater-processing Chlorella strains: Chlorella sorokiniana BD09, Chlorella sorokiniana BD08 and Chlorella sp. Dachan, and the previously published Chlorella sorokiniana UTEX 1602, showing us that BD09 maintains the best tolerance in synthetic wastewater. We then performed genome sequencing and analysis, resulting in a high-quality assembly for each genome with scaffold N50 > 2 Mb and genomic completeness ≥91%, as well as genome annotation with 9,668, 10,240, 9,821 high-confidence gene models predicted for BD09, BD08, and Dachan, respectively. Comparative genomics study unravels that metabolic pathways, which are involved in nitrogen and phosphorus assimilation, were enriched in the faster-growing strains. We found that gene structural variation and genomic rearrangement might contribute to differential capabilities in wastewater tolerance among the strains, as indicated by gene copy number variation, domain reshuffling of orthologs involved, as well as a ~1 Mb-length chromosomal inversion we observed in BD08 and Dachan. In addition, we speculated that an associated bacterium, Microbacterium chocolatum, which was identified within Dachan, play a possible role in synergizing nutrient removal. Our three newly sequenced Chlorella genomes provide a fundamental foundation to understand the molecular basis of abiotic stress tolerance in wastewater treatment, which is essential for future genetic engineering and strain improvement.

Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii.

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.

Characterization and expression profiles of small heat shock proteins in the marine red alga Pyropia yezoensis.

Small heat shock proteins (sHSPs) are found in all three domains of life (Bacteria, Archaea, and Eukarya) and play a critical role in protecting organisms from a range of environmental stresses. However, little is known about their physiological functions in red algae. Therefore, we characterized the sHSPs (PysHSPs) in the red macroalga Pyropia yezoensis, which inhabits the upper intertidal zone where it experiences fluctuating stressful environmental conditions on a daily and seasonal basis, and examined their expression profiles at different developmental stages and under varying environmental conditions. We identified five PysHSPs (PysHSP18.8, 19.1, 19.2, 19.5, and 25.8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that expression of the genes PysHSP18.8, PysHSP19.5, and PysHSP25.8 was repressed at all the developmental stages under normal conditions, whereas PysHSP19.1 and PysHSP19.2 were overexpressed in mature gametophytes and sporophytes. Exposure of the gametophytes to high temperature, oxidative stress, or copper significantly increased the mRNA transcript levels of all the five genes, while exogenous application of the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) significantly increased the expression levels of PysHSP19.2, PysHSP19.5, and PysHSP25.8. These findings will help to further our understanding of the role of PysHSP genes and provide clues about how Pyropia species can adapt to the stressful conditions encountered in the upper intertidal zone during their life cycle.

Cloning and transcription analysis of the nitrate reductase gene from Haematococcus pluvialis.

OBJECTIVES: To optimize the cultivation media for the growth rate of Haematococcus pluvialis and to study the transcription regulation of the algal nitrate reductase (NR), a key enzyme for nitrogen metabolism. RESULTS: The NR gene from H. pluvialis hd7 consists of 5636 nucleotides, including 14 introns. The cDNA ORF is 2718 bp, encoding a 905 aa protein with three conserved domains. The NR amino acids of H. pluvialis hd7 are hydrophilic and have similarity of 72% compared to that of Dunaliella. NR transcription increased with an increase of nitrate concentration from 0.4 to 1 g/l. A deficiency of nitrogen increased NR transcription significantly. The transcription level of NR increased at phosphorus concentrations from 0.08 to 0.2 g/l, with a maximum at 0.08 g/l. The optimum parameters of medium component for transcription of NR and growth of H. pluvialis were 0.3 g NaNO3/l, 0.045 g KH2PO4/l and 1.08 g sodium acetate/l. CONCLUSIONS: This study provides a better understanding of nitrate regulation in H. pluvialis.

Growth on ATP Elicits a P-Stress Response in the Picoeukaryote Micromonas pusilla.

The surface waters of oligotrophic oceans have chronically low phosphate (Pi) concentrations, which renders dissolved organic phosphorus (DOP) an important nutrient source. In the subtropical North Atlantic, cyanobacteria are often numerically dominant, but picoeukaryotes can dominate autotrophic biomass and productivity making them important contributors to the ocean carbon cycle. Despite their importance, little is known regarding the metabolic response of picoeukaryotes to changes in phosphorus (P) source and availability. To understand the molecular mechanisms that regulate P utilization in oligotrophic environments, we evaluated transcriptomes of the picoeukaryote Micromonas pusilla grown under Pi-replete and -deficient conditions, with an additional investigation of growth on DOP in replete conditions. Genes that function in sulfolipid substitution and Pi uptake increased in expression with Pi-deficiency, suggesting cells were reallocating cellular P and increasing P acquisition capabilities. Pi-deficient M. pusilla cells also increased alkaline phosphatase activity and reduced their cellular P content. Cells grown with DOP were able to maintain relatively high growth rates, however the transcriptomic response was more similar to the Pi-deficient response than that seen in cells grown under Pi-replete conditions. The results demonstrate that not all P sources are the same for growth; while M. pusilla, a model picoeukaryote, may grow well on DOP, the metabolic demand is greater than growth on Pi. These findings provide insight into the cellular strategies which may be used to support growth in a stratified future ocean predicted to favor picoeukaryotes.

Isolation and characterization of a mutant defective in triacylglycerol accumulation in nitrogen-starved Chlamydomonas reinhardtii.

Triacylglycerol (TAG), a major source of biodiesel production, accumulates in nitrogen-starved Chlamydomonas reinhardtii. However, the metabolic pathway of starch-to-TAG conversion remains elusive because an enzyme that affects the starch degradation is unknown. Here, we isolated a new class of mutant bgal1, which expressed an overaccumulation of starch granules and defective photosynthetic growth. The bgal1 was a null mutant of a previously uncharacterized ß-galactosidase-like gene (Cre02.g119700), which decreased total ß-galactosidase activity 40% of the wild type. Upon nitrogen starvation, the bgal1 mutant showed decreased TAG accumulation mainly due to the reduced flux of de novo TAG biosynthesis evidenced by increased unsaturation of fatty acid composition in TAG and reduced TAG accumulation by additional supplementation of acetate to the culture media. Metabolomic analysis of the bgal1 mutant showed significantly reduced levels of metabolites following the hydrolysis of starch and substrates for TAG accumulation, whereas metabolites in TCA cycle were unaffected. Upon nitrogen starvation, while levels of glucose 6-phosphate, fructose 6-phosphate and acetyl-CoA remained lower, most of the other metabolites in glycolysis were increased but those in the TCA cycle were decreased, supporting TAG accumulation. We suggest that BGAL1 may be involved in the degradation of starch, which affects TAG accumulation in nitrogen-starved C. reinhardtii. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

Characterization and expression of AMP-forming Acetyl-CoA Synthetase from Dunaliella tertiolecta and its response to nitrogen starvation stress.

AMP-forming acetyl-CoA synthetase (ACS) catalyzes the formation of acetyl-CoA. Here, a cDNA of ACS from Dunaliella tertiolecta (DtACS) was isolated using RACEs. The full-length DtACS cDNA (GenBank: KT692941) is 2,464 bp with a putative ORF of 2,184 bp, which encodes 727 amino acids with a predicted molecular weight of 79.72 kDa. DtACS has a close relationship with Chlamydomonas reinhardtii and Volvox carteri f. nagariensis. ACSs existing in Bacteria, Archaea and Eukaryota share ten conserved motifs (A1-A10) and three signature motifs (I-III) of the acyl-adenylate/thioester forming enzyme superfamily. DtACS was expressed in E. coli BL21 as Trx-His-tagged fusion protein (~100 kDa) and the enzymatic activity was detected. The recombinant DtACS was purified by HisTrap(TM) HP affinity chromatography to obtain a specific activity of 52.873 U/mg with a yield of 56.26%, which approached the specific activity of ACS isolated from other eukaryotes. Kinetic analysis indicated that the Km of DtACS was 3.59 mM for potassium acetate, and the purified DtACS exhibited a temperature optimum of 37 °C and a pH optimum of 8.0. In addition, the expression levels of DtACS were increased after nitrogen starvation cultivation, indicating that ACS activity may be related to the lipid accumulation under nitrogen deficient condition.

Biochemical Modulation by Carbon and Nitrogen Addition in Cultures of Dictyota menstrualis (Dictyotales, Phaeophyceae) to Generate Oil-based Bioproducts.

Dictyota menstrualis (Hoyt) Schnetter, Hörning & Weber-Peukert (Dictyotales, Phaeophyceae) was studied for the production of oil-based bioproducts and co-products. Experiments were performed to evaluate the effect of carbon dioxide (CO2) concentration, under nitrogen (NO3 (-)) limiting and saturation conditions, on growth rate (GR), photosynthesis, as well as nitrate reductase (NR), carbonic anhydrase (CA), and Rubisco activities. In addition, the biochemical composition of D. menstrualis under these conditions was estimated. GR, protein content, and N content in D. menstrualis were higher in treatments containing NO3 (-), irrespective of CO2 addition. However, when CO2 was added to medium saturated with NO3 (-), values of maximum photosynthesis, Rubisco, and NR activity, as well as total soluble carbohydrates and lipids, were increased. CA activity did not vary under the different treatments. The fatty acid profile of D. menstrualis was characterized by a high content of polyunsaturated fatty acids, especially the omega-3 fatty acids, making it a possible candidate for nutraceutical use. In addition, this species presented high GR, photosynthetic rate, and fatty acid content, highlighting its economic importance and the possibility of different biotechnological applications.

Relationship of proteomic variation and toxin synthesis in the dinoflagellate Alexandrium tamarense CI01 under phosphorus and inorganic nitrogen limitation.

Paralytic shellfish toxins (PSTs) are originated from cyanobacteria and dinoflagellates, including Alexandrium tamarense, the common dinoflagellate species. In this study, a toxic dinoflagellate strain of A. tamarense CI01 was selected for studying the PSTs' concentration and the related protein variation during the whole cell cycle under different nutrient conditions. High-performance liquid chromatography, 2-D DIGE and Western blotting were used collectively for protein profiling and identification. Results showed that the toxin content was suppressed under nitrogen limiting condition, but enhanced in phosphorous limiting medium. Based on the results of proteomics analysis, 7 proteins were discovered to be related to the PSTs biosynthesis of A. tamarense CI01, including S-adenosylhomocysteine hydrolase, ornithine cyclodeaminase, argininosuccinate synthase, methyluridine methyltransferase cystine ABC transporter, phosphoserine phosphatase, argininosuccinate synthase and acyl-CoA dehydrogenase, which corresponds to the metabolism of the methionine, cysteine, ornithine, arginine and proline. Moreover, some photosynthesis relating proteins also increased their expression during PST synthesis period in A. tamarense CI01, such as phosphoenolpyruvate carboxylase, chloroplast phosphoglycerate kinase, peridinin-chlorophyll α-binding protein, Mg(2+) transporter protein and chloroplast phosphoglycerate kinase. The above findings are in support of our hypothesis that these proteins are involved in toxin biosynthesis of A. tamarense CI01, but cause-and-effect mechanisms need to be investigated in further studies.

Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt.

Astaxanthin is a highly valued carotenoid with strong antioxidant activity and has wide applications in aquaculture, food, cosmetic, and pharmaceutical industries. The market demand for natural astaxanthin promotes research in metabolic engineering of heterologous hosts for astaxanthin production. In this study, an astaxanthin-producing Saccharomyces cerevisiae strain was created by successively introducing the Haematococcus pluvialis ß-carotenoid hydroxylase (crtZ) and ketolase (bkt) genes into a previously constructed ß-carotene hyperproducer. Further integration of strategies including codon optimization, gene copy number adjustment, and iron cofactor supplementation led to significant increase in the astaxanthin production, reaching up to 4.7 mg/g DCW in the shake-flask cultures which is the highest astaxanthin content in S. cerevisiae reported to date. Besides, the substrate specificity of H. pluvialis CrtZ and BKT and the probable formation route of astaxanthin from ß-carotene in S. cerevisiae were figured out by expressing the genes separately and in combination. The yeast strains engineered in this work provide a basis for further improving biotechnological production of astaxanthin and might offer a useful general approach to the construction of heterologous biosynthetic pathways for other natural products.

Expression of bkt and bch genes from Haematococcus pluvialis in transgenic Chlamydomonas.

ß-carotene ketolase and ß-carotene hydroxylase encoded by bkt and bch, respectively, are key enzymes required for astaxanthin biosynthesis in Haematococcu pluvialis 34-1n. Two expression vectors containing cDNA sequences of bkt and bch were constructed and co-transformed into cell-wall-deficient Chlamydomonas reinhardtii CC-849. Transgenic algae were screened on TAP agar plates containing 10 µg mL(-1) Zeomycin. PCR-Southern analysis showed that bkt and bch were integrated into the genomes of C. reinhardtii. Transcripts of bkt and bch were further confirmed by RT-PCR-Southern analysis. Compared with the wild type, transgenic algae produced 29.04% and 30.27% more carotenoids and xanthophylls, respectively. Moreover, the transgenic algae could accumulate 34% more astaxanthin than wild type. These results indicate that foreign bkt and bch genes were successfully translated into ß-carotene ketolase and ß-carotene hydroxylase, which were responsible for catalyzing the biosynthesis of astaxanthin in transgenic algae.

Intracellular localization and induction of a dynamic RNA-editing event of macro-algal V-ATPase subunit A (VHA-A) in response to copper.

A V-ATPase subunit A protein (VHA-A) transcript together with a variant (C793 to U), which introduces a stop codon truncating the subunit immediately downstream of its ATP binding site, was identified within a Fucus vesiculosus cDNA from a heavy metal contaminated site. This is intriguing because the VHA-A subunit is the crucial catalytic subunit responsible for the hydrolysis of ATP that drives ion transport underlying heavy metal detoxification pathways. We employed a chemiluminescent hybridization protection assay to quantify the proportion of both variants directly from mRNA while performing quantification of total transcript using Q-PCR. Polyclonal antisera raised against recombinant VHA-A facilitated simultaneous detection of parent and truncated VHA-A and revealed its cellular and subcellular localization. By exploiting laboratory exposures and samples from an environmental copper gradient, we showed that total VHA-A transcript and protein, together with levels of the truncated variant, were induced by copper. The absence of a genomic sequence representing the truncated variant suggests a RNA editing event causing the production of the truncated VHA-A. Based on these observations, we propose RNA editing as a novel molecular process underpinning VHA trafficking and intracellular sequestration of heavy metals under stress.

Expression of a xanthine permease and phosphate transporter in cultures and field populations of the harmful alga Aureococcus anophagefferens: tracking nutritional deficiency during brown tides.

Targeted gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to track patterns in the expression of genes indicative of nitrogen or phosphorus deficiency in the brown tide-forming alga Aureococcus anophagefferens. During culture experiments, a xanthine/uracil/vitamin C permease (XUV) was upregulated ∼20-fold under nitrogen-deficient conditions relative to a nitrogen-replete control and rapidly returned to nitrogen-replete levels after nitrogen-deficient cells were resupplied with nitrate or ammonium. It was not responsive to phosphorus deficiency. Expression of an inorganic phosphate transporter (PTA3) was enriched ∼10-fold under phosphorus-deficient conditions relative to a phosphorus-replete control, and this signal was rapidly lost upon phosphate resupply. PTA3 was not upregulated by nitrogen deficiency. Natural A. anophagefferens populations from a dense brown tide that occurred in Long Island, NY, in 2009 were assayed for XUV and PTA3 expression and compared with nutrient concentrations over the peak of a bloom. Patterns in XUV expression were consistent with nitrogen-replete growth, never reaching the values observed in N-deficient cultures. PTA3 expression was highest prior to peak bloom stages, reaching expression levels within the range of P-deficient cultures. These data highlight the value of molecular-level assessments of nutrient deficiency and suggest that phosphorus deficiency could play a role in the dynamics of destructive A. anophagefferens blooms.

Photoprotection in the green tidal alga Ulva prolifera: role of LHCSR and PsbS proteins in response to high light stress.

Ulva prolifera, an intertidal macroalga, has to adapt to wide variations in light intensity, making this species particularly rewarding for studying the evolution of photoprotective mechanisms. Intense light induced increased non-photochemical quenching (NPQ) and stimulated de-epoxidation of xanthophyll cycle components, while DTT-treated samples had lower NPQ capacity, indicating that the xanthophyll cycle must participate in photoprotection. In this work, we found that the PsbS-related NPQ was maintained in U. prolifera. According to analysed gene expression, both LhcSR and psbS were up-regulated in high light, suggesting that these two genes are light-induced. LHCSR and PsbS proteins were present at different light intensities and accumulated under high light conditions, and PsbS concentrations were higher than LHCSR, showing that the NPQ mechanism of U. prolifera is more dependent on PsbS protein concentration. Moreover, the level of both LHCSR and PsbS proteins was high even in the darkness, and neither the transcript level nor protein content of LhcSR and psbS genes varied significantly following short-term exposure to intense light. These findings suggest that this alga can modulate NPQ levels through regulation of the xanthophyll cycle and concentrations of PsbS and/or LHCSR.

Photosynthetic circadian rhythmicity patterns of Symbiodinium, [corrected] the coral endosymbiotic algae.

Biological clocks are self-sustained endogenous timers that enable organisms (from cyanobacteria to humans) to anticipate daily environmental rhythms, and adjust their physiology and behaviour accordingly. Symbiotic corals play a central role in the creation of biologically rich ecosystems based on mutualistic symbioses between the invertebrate coral and dinoflagellate protists from the genus Symbiodinium. In this study, we experimentally establish that Symbiodinium photosynthesis, both as a free-living unicellular algae and as part of the symbiotic association with the coral Stylophora pistillata, is 'wired' to the circadian clock mechanism with a 'free-run' cycle close to 24 h. Associated photosynthetic pigments also showed rhythmicity under light/dark conditions and under constant light conditions, while the expression of the oxygen-evolving enhancer 1 gene (within photosystem II) coincided with photosynthetically evolved oxygen in Symbiodinium cultures. Thus, circadian regulation of the Symbiodinium photosynthesis is, however, complicated as being linked to the coral/host that have probably profound physiochemical influence on the intracellular environment. The temporal patterns of photosynthesis demonstrated here highlight the physiological complexity and interdependence of the algae circadian clock associated in this symbiosis and the plasticity of algae regulatory mechanisms downstream of the circadian clock.

Cloning and selection of carotenoid ketolase genes for the engineering of high-yield astaxanthin in plants.

ß-Carotene ketolase (BKT) catalyzes the rate-limiting steps for the biosynthesis of astaxanthin. Several bkt genes have been isolated and explored to modify plant carotenoids to astaxanthin with limited success. In this study, five algal BKT cDNAs were isolated and characterized for the engineering of high-yield astaxanthin in plants. The products of the cDNAs showed high similarity in sequence and enzymatic activity of converting ß-carotene into canthaxanthin. However, the enzymes exhibited extremely different activities in converting zeaxanthin into astaxanthin. Chlamydomonas reinhardtii BKT showed the highest conversion rate (ca 85%), whereas, Neochloris wimmeri BKT exhibited very poor activity of ketolating zeaxanthin. Expression of C. reinhardtii BKT in tobacco led to a twofold increase of total carotenoids in the leaves with astaxanthin being the predominant. The bkt genes described here provide a valuable resource for metabolic engineering of plants as cell factories for astaxanthin production.

Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1.

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.