l e t t e r sHow an insect evolves to become a successful herbivore is of profound biological and practical importance. Herbivores are often adapted to feed on a specific group of evolutionarily and biochemically related host plants 1 , but the genetic and molecular bases for adaptation to plant defense compounds remain poorly understood 2 . We report the first whole-genome sequence of a basal lepidopteran species, Plutella xylostella, which contains 18,071 protein-coding and 1,412 unique genes with an expansion of gene families associated with perception and the detoxification of plant defense compounds. A recent expansion of retrotransposons near detoxification-related genes and a wider system used in the metabolism of plant defense compounds are shown to also be involved in the development of insecticide resistance. This work shows the genetic and molecular bases for the evolutionary success of this worldwide herbivore and offers wider insights into insect adaptation to plant feeding, as well as opening avenues for more sustainable pest management.The global pest P. xylostella (Lepidoptera: Yponomeutidae) is thought to have coevolved with the crucifer plant family 3 ( Supplementary Fig. 1) and has become the most destructive pest of economically important food crops, including rapeseed, cauliflower and cabbage 4 . Recently, the total cost of damage and management worldwide was estimated at $4-5 billion per annum 5,6 . This insect is the first species to have evolved resistance to dichlorodiphenyltrichloroethane (DDT) in the 1950s 7 and to Bacillus thuringiensis (Bt) toxins in the 1990s 8 and has developed resistance to all classes of insecticide, making it increasingly difficult to control 9,10 . P. xylostella provides an exceptional system for understanding the genetic and molecular bases of how insect herbivores cope with the broad range of plant defenses and chemicals encountered in the environment (Supplementary Fig. 2).We used a P. xylostella strain (Fuzhou-S) collected from a field in Fuzhou in southeastern China (26.08 °N, 119.28 °E) for sequencing ( Supplementary Fig. 1). Whole-genome shotgun-based Illumina sequencing of single individuals (Supplementary Table 1), even after ten generations of laboratory inbreeding, resulted in a poor initial assembly (N50 = 2.4 kb, representing the size above which 50% of the total length of the sequences is included), owing to high levels of heterozygosity ( Supplementary Figs. 3 and 4 and Supplementary Table 2). Subsequently, we sequenced 100,800 fosmid clones (comprising ~10× the genome length) to a depth of 200× ( Supplementary Fig. 5 and Supplementary Tables 3-5), assembling the resulting sequence data into 1,819 scaffolds, with an N50 of 737 kb, spanning ~394 Mb of the genome sequence (version 1; Supplementary Fig. 6 and Supplementary Table 6). The assembly covered 85.5% of a set of protein-coding ESTs (Supplementary Tables 7 and 8) generated by transcriptome sequencing 11 . Alignment of a subject scaffold against a 126-kb BAC (GenBank GU058050) from an altern...
It has been widely accepted that the primary function of the Lands cycle is to provide a route for acyl remodeling to modify fatty acid (FA) composition of phospholipids derived from the Kennedy pathway. Lysophosphatidylcholine acyltransferase (LPCAT) is an evolutionarily conserved key enzyme in the Lands cycle. In this study, we provide direct evidence that the Arabidopsis thaliana LPCATs, LPCAT1 and LPCAT2, participate in the Lands cycle in developing seeds. In spite of a substantially reduced initial rate of nascent FA incorporation into phosphatidylcholine (PC), the PC level in the double mutant lpcat1 lpcat2-2 remained unchanged. LPCAT deficiency triggered a compensatory response of de novo PC synthesis and a concomitant acceleration of PC turnover that were attributable at least in part to PC deacylation. Acyl-CoA profile analysis revealed complicated metabolic alterations rather than merely reduced acyl group shuffling from PC in the mutant. Shifts in FA stereospecific distribution in triacylglycerol of the mutant seed suggested a preferential retention of saturated acyl chains at the stereospecific numbering (sn)-1 position from PC and likely a channeling of lysophosphatidic acid, derived from PC, into the Kennedy pathway. Our study thus illustrates an intricate relationship between the Lands cycle and the Kennedy pathway.
Clubroot, caused by Plasmodiophora brassicae, is an important disease on Brassica species worldwide. A clubroot resistance gene, Rcr1, with efficacy against pathotype 3 of P. brassicae, was previously mapped to chromosome A03 of B. rapa in pak choy cultivar “Flower Nabana”. In the current study, resistance to pathotypes 2, 5 and 6 was shown to be associated with Rcr1 region on chromosome A03. Bulked segregant RNA sequencing was performed and short read sequences were assembled into 10 chromosomes of the B. rapa reference genome v1.5. For the resistant (R) bulks, a total of 351.8 million (M) sequences, 30,836.5 million bases (Mb) in length, produced 120-fold coverage of the reference genome. For the susceptible (S) bulks, 322.9 M sequences, 28,216.6 Mb in length, produced 109-fold coverage. In total, 776.2 K single nucleotide polymorphisms (SNPs) and 122.2 K insertion / deletion (InDels) in R bulks and 762.8 K SNPs and 118.7 K InDels in S bulks were identified; each chromosome had about 87% SNPs and 13% InDels, with 78% monomorphic and 22% polymorphic variants between the R and S bulks. Polymorphic variants on each chromosome were usually below 23%, but made up 34% of the variants on chromosome A03. There were 35 genes annotated in the Rcr1 target region and variants were identified in 21 genes. The numbers of poly variants differed significantly among the genes. Four out of them encode Toll-Interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat proteins; Bra019409 and Bra019410 harbored the higher numbers of polymorphic variants, which indicates that they are more likely candidates of Rcr1. Fourteen SNP markers in the target region were genotyped using the Kompetitive Allele Specific PCR method and were confirmed to associate with Rcr1. Selected SNP markers were analyzed with 26 recombinants obtained from a segregating population consisting of 1587 plants, indicating that they were completely linked to Rcr1. Nine SNP markers were used for marker-assisted introgression of Rcr1 into B. napus canola from B. rapa, with 100% accuracy in this study.
<b><i>Background:</i></b> Levels of urinary microvesicles, which are increased during various kidney injuries, have diagnostic potential for renal diseases. However, the significance of urinary microvesicles as a renal disease indicator is dampened by the difficulty to ascertain their cell source. <b><i>Objectives:</i></b> The aim of this study was to demonstrate that podocytes can release migrasomes, a unique class of microvesicle with size ranging between 400 and 2,000 nm, and the urine level of migrasomes may serve as novel non-invasive biomarker for early podocyte injury. <b><i>Method:</i></b> In this study, immunofluorescence labeling, electronic microscopy, nanosite, and sequential centrifugation were used to purify and analyze migrasomes. <b><i>Results:</i></b> Migrasomes released by podocytes differ from exosomes as they have different content and mechanism of release. Compared to podocytes, renal tubular cells secrete markedly less migrasomes. Moreover, secretion of migrasomes by human or murine podocytes was strongly augmented during podocyte injuries induced by LPS, puromycin amino nucleoside (PAN), or a high concentration of glucose (HG). LPS, PAN, or HG-induced podocyte migrasome release, however, was blocked by Rac-1 inhibitor. Strikingly, a higher level of podocyte migrasomes in urine was detected in mice with PAN-nephropathy than in control mice. In fact, increased urinary migrasome number was detected earlier than elevated proteinuria during PAN-nephropathy, suggesting that urinary migrasomes are a more sensitive podocyte injury indicator than proteinuria. Increased urinary migrasome number was also detected in diabetic nephropathy patients with proteinuria level <5.5 g/day. <b><i>Conclusions:</i></b> Our findings reveal that podocytes release the “injury-related” migrasomes during migration and provide urinary podocyte migrasome as a potential diagnostic marker for early podocyte injury.
Cellular phospholipids undergo deacylation and reacylation through a process known as Lands cycle. In this report, we provide evidence demonstrating that yeast YOR175c, herein designated as LCA1, encodes a key component of the Lands cycle, the acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT). Deletion of LCA1 resulted in a drastic reduction in LPCAT activity, while over expression led to a several fold increase in enzyme activity. We further show that disruption of LCA1 caused an enhanced production of glycerophosphorylcholine, a product of phosphatidylcholine (PC) deacylation and that the lysophosphatidic acid acyltransferase SLC1 was not involved in this process. Identification of LCA1 provides an essential molecular tool for further study of Lands cycle in PC turnover.
Fatty acyl esters of phytosterols are a major form of sterol conjugates distributed in many parts of plants. In this study we report an Arabidopsis (Arabidopsis thaliana) gene, AtSAT1 (At3g51970), which encodes for a novel sterol O-acyltransferase. When expressed in yeast (Saccharomyces cerevisiae), AtSAT1 mediated production of sterol esters enriched with lanosterol. Enzyme property assessment using cell-free lysate of yeast expressing AtSAT1 suggested the enzyme preferred cycloartenol as acyl acceptor and saturated fatty acyl-Coenyzme A as acyl donor. Taking a transgenic approach, we showed that Arabidopsis seeds overexpressing AtSAT1 accumulated fatty acyl esters of cycloartenol, accompanied by substantial decreases in ester content of campesterol and b-sitosterol. Furthermore, fatty acid components of sterol esters from the transgenic lines were enriched with saturated and long-chain fatty acids. The enhanced AtSAT1 expression resulted in decreased level of free sterols, but the total sterol content in the transgenic seeds increased by up to 60% compared to that in wild type. We conclude that AtSAT1 mediates phytosterol ester biosynthesis, alternative to the route previously described for phospholipid:sterol acyltransferase, and provides the molecular basis for modification of phytosterol ester level in seeds.
Genetic resistance is widely used to manage clubroot (Plasmodiophora brassicae) in brassica crops, but new pathotypes have recently been identified on canola (Brassica napus) on the Canadian prairies. Resistance effective against both the most prevalent pathotype (3H, based on the Canadian Clubroot Differential system) and the new pathotypes is needed. BC1 plants of Brassica rapa from a cross of line 96-6990-2 (clubroot resistance originating from turnip cultivar ‘Waaslander’) and a susceptible doubled-haploid line, ACDC, exhibited a 1:1 segregation for resistance against pathotypes 3H and 5X. A resistance gene designated as Rcr3 was mapped initially based on the percentage of polymorphic variants using bulked segregant RNA sequencing (BSR-Seq) and further mapped using Kompetitive Allele Specific PCR. DNA variants were identified by assembling short reads against a reference genome of B. rapa. Rcr3 was mapped into chromosome A08. It was flanked by single nucleotide polymorphisms (SNP) markers (A90_A08_SNP_M12 and M16) between 10.00 and 10.23 Mb, in an interval of 231.6 Kb. There were 32 genes in the Rcr3 interval. Three genes (Bra020951, Bra020974, and Bra020979) were annotated with disease resistance mechanisms, which are potential candidates for Rcr3. Another resistance gene, designated as Rcr9wa, for resistance to pathotype 5X was mapped, with the flanking markers (A90_A08_SNP_M28 and M79) between 10.85 and 11.17 Mb using the SNP sites identified through BSR-Seq for Rcr3. There were 44 genes in the Rcr9wa interval, three of which (Bra020827, Bra020828, Bra020814) were annotated as immune-system-process related genes, which are potential candidates for Rcr9wa.
An important enzyme involved in phospholipid turnover is the acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT). Here, we report characterization of a newly discovered human LPCAT (LPCAT3), which has distinct substrate preferences strikingly consistent with a role in phosphatidylcholine (PtdCho) remodeling and modulating fatty acid composition of PtdCho. LPCAT3 prefers lysophosphatidylcholine (lysoPtdCho) with saturated fatty acid at the sn-1 position and exhibits acyl donor preference towards linoleoyl-CoA and arachidonoyl-CoA. Furthermore, LPCAT3 is active in mediating 1-O-alkyl-sn-glycero-3-phosphocholine acylation with long chain fatty acyl-CoAs to generate 1-O-alkyl-phosphatidylcholine, another very important constitute of mammalian membrane systems. These properties are precisely the known attributes of LPCAT previously ascribed to the isoform involved in Lands' cycle, and thus strongly suggest that LPCAT3 is involved in phospholipids remodeling to achieve appropriate membrane lipid fatty acid composition.
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