a-caenorhabditis-elegans-model-system-for-amylopathy-study

A <em>Caenorhabditis elegans</em> Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

University of Medicine and Dentistry of New Jersey

Entomopathogenic fungi such as Metarhizium anisopliae infect insects by direct penetration of the cuticle, after which the fungus adapts to the high osmotic pressure of the hemolymph and multiplies. Here we characterize the M. anisopliae Mos1 gene and demonstrate that it encodes the osmosensor required for this process. MOS1 contains transmembrane regions and a C-terminal Src homology 3 domain similar to those of yeast osmotic adaptor proteins, and homologs of MOS1 are widely distributed in the fungal kingdom. Reverse transcription-PCR demonstrated that Mos1 is up-regulated in insect hemolymph as well as artificial media with high osmotic pressure. Transformants containing an antisense vector directed to the Mos1 mRNA depleted transcript levels by 80%. This produced selective alterations in regulation of genes involved in hyphal body formation, cell membrane stiffness, and generation of intracellular turgor pressure, suggesting that these processes are mediated by MOS1. Consistent with a role in stress responses, transcript depletion of Mos1 increased sensitivity to osmotic and oxidative stresses and to compounds that interfere with cell wall biosynthesis. It also disrupted developmental processes, including formation of appressoria and hyphal bodies. Insect bioassays confirmed that Mos1 knockdown significantly reduces virulence. Overall, our data show that M. anisopliae MOS1 mediates cellular responses to high osmotic pressure and subsequent adaptations to colonize host hemolymph.

MOS1 osmosensor of Metarhizium anisopliae is required for adaptation to insect host hemolymph.

Champignons entomopathog&egrave;nes comme Metarhizium anisopliae infectent des insectes par p&eacute;n&eacute;tration directe de la cuticule, apr&egrave;s quoi le champignon s&#39;adapte &agrave; la forte pression osmotique de l&#39;h&eacute;molymphe et se multiplie. Ici, nous caract&eacute;risons la g&egrave;ne de M. anisopliae Mos1 et d&eacute;montrer qu&#39;il encode l&#39;osmosensor n&eacute;cessaire &agrave; ce processus. Mos1 contient des r&eacute;gions transmembranaires et un domaine d&#39;homologie 3 C-terminal Src semblable &agrave; celles des prot&eacute;ines de levure adaptateur osmotique et homologues de MOS1 sont largement distribu&eacute;s dans le Royaume Champignon. Reverse transcription-PCR a d&eacute;montr&eacute; que Mos1 est r&eacute;gul&eacute;e dans l&#39;h&eacute;molymphe insecte ainsi que des milieux artificiels avec forte pression osmotique. Transformants contenant un vecteur antisens dirig&eacute; vers l&#39;ARNm Mos1 appauvri niveaux de transcription de 80 %. Ceci a produit des alt&eacute;rations dans la r&eacute;gulation des g&egrave;nes impliqu&eacute;s dans la formation de corps d&#39;hyphes, rigidit&eacute; de la membrane cellulaire et la g&eacute;n&eacute;ration de la pression de turgescence intracellulaire, ce qui sugg&egrave;re que ces processus sont m&eacute;di&eacute;s par MOS1 s&eacute;lectives. Compatible avec un r&ocirc;le dans les r&eacute;actions de stress, &eacute;puisement de transcription de Mos1 augmente la sensibilit&eacute; aux stress osmotiques et oxydatifs et aux compos&eacute;s qui interf&egrave;rent avec la biosynth&egrave;se de la paroi cellulaire. Il &eacute;galement perturb&eacute; des processus de d&eacute;veloppement, y compris la formation des appressoria et corps myc&eacute;liens. Insectes bio-essais ont confirm&eacute; que Mos1 knockdown r&eacute;duit sensiblement virulence. Dans l&#39;ensemble, nos r&eacute;sultats montrent que M. anisopliae MOS1 intervient dans la r&eacute;ponse cellulaire &agrave; forte pression osmotique et les adaptations ult&eacute;rieures &agrave; coloniser l&#39;h&eacute;molymphe de l&#39;h&ocirc;te.

Mos1 osmosensor de Metarhizium anisopliae est n&eacute;cessaire pour l&#39;adaptation de l&#39;h&eacute;molymphe de l&#39;insecte h&ocirc;te.

Funghi entomopatogeni quali Metarhizium anisopliae infettano gli insetti da diretta penetrazione della cuticola, dopo che il fungo si adatta all&#39;alta pressione osmotica dell&#39;emolinfa e moltiplica. Qui noi caratterizzare il gene di M. anisopliae Mos1 e dimostrare che codifica per la osmosensor necessaria per questo processo. MOS1 contiene regioni transmembrane e un dominio di omologia 3 C-terminale Src simile a quelli delle proteine di lievito adattatore osmotico e omologhi di MOS1 sono ampiamente distribuiti nel Regno fungoso. Trascrizione inversa-PCR ha dimostrato che Mos1 &egrave; up-regolato nell&#39;emolinfa degli insetti cos&igrave; come mezzi artificiali con alta pressione osmotica. Transformants contenente un vettore antisenso diretto a mRNA Mos1 impoverito livelli di trascrizione del 80%. Questo prodotto selettivi alterazioni nella regolazione dei geni coinvolti nella formazione del corpo ifale, rigidit&agrave; della membrana cellulare e generazione di turgescenza intracellulare, suggerendo che questi processi sono mediati da MOS1. Coerente con un ruolo nelle risposte di stress, esaurimento di trascrizione di Mos1 aumentata sensibilit&agrave; a stress osmotico e ossidativo e ai composti che interferiscono con la biosintesi della parete cellulare. Esso perturbato anche processi di sviluppo, compresa la formazione di appressoria e corpi ifali. Analisi biologiche dell&#39;insetto ha confermato che Mos1 atterramento riduce significativamente la virulenza. Nel complesso, i nostri dati mostrano che M. anisopliae MOS1 da intermediario cellular responses to ad alta pressione osmotica e successivi adattamenti di colonizzare l&#39;emolinfa host.

MOS1 osmosensor di Metarhizium anisopliae &egrave; necessaria per l&#39;adeguamento al emolinfa degli insetti host.

Pentose metabolism through the phosphoketolase pathway has been well characterized in bacteria. In this paper, we report the identification of a phosphoketolase homologue Mpk1 in the insect-pathogenic fungus Metarhizium anisopliae. Phylogenetic analysis showed that fungal phosphoketolases are of bacterial origin and diverged into two superfamilies. Frequent gene loss or lack of acquisition is evident in specific fungal lineages or species. The mpk1 gene is highly expressed when grown in trehalose-rich insect haemolymph but poorly induced by insect cuticle or carbohydrate-rich plant root exudate. In addition, mpk1 gene expression and enzyme activity could be upregulated by different sugars including xylose, trehalose, glucose or sucrose. mpk1 null mutants generated by homologous recombination grew similar to the wild type of M. anisopliae on medium amended with xylose as a sole carbon source. However, insect (tobacco hornworm, Manduca sexta) bioassays showed significantly reduced virulence in Deltampk1. The results of this study suggest that the horizontally transferred Mpk1 in M. anisopliae plays an important niche adaptation role for fungal propagation in insect haemocoel. Following the carbohydrate flux from plants to plant-feeding insects and insect pathogenic fungi, a tritrophic relationship is discussed in association with the requirement of fungal phosphoketolase pathway.

A phosphoketolase Mpk1 of bacterial origin is adaptively required for full virulence in the insect-pathogenic fungus Metarhizium anisopliae.

M&eacute;tabolisme de pentose par la voie de phosphoketolase a &eacute;t&eacute; bien caract&eacute;ris&eacute; dans les bact&eacute;ries. Dans cet article, nous rapportons l&#39;identification d&#39;un homologue de phosphoketolase Mpk1 dans l&#39;insectes pathog&egrave;nes champignon Metarhizium anisopliae. L&#39;analyse phylog&eacute;n&eacute;tique a montr&eacute; que phosphoketolases fongiques sont d&#39;origine bact&eacute;rienne et diverg&eacute; en deux superfamilles. Perte de g&egrave;nes fr&eacute;quentes ou de manque d&#39;acquisition est &eacute;vident dans des lign&eacute;es sp&eacute;cifiques fongiques ou esp&egrave;ces. Le g&egrave;ne mpk1 est fortement exprim&eacute; lorsque cultiv&eacute;es dans l&#39;h&eacute;molymphe des insecte riches en tr&eacute;halose mais mal induite par la cuticule insecte ou exsudat racinaire v&eacute;g&eacute;taux riches en hydrates de carbone. En outre, l&#39;activit&eacute; enzymatique et de l&#39;expression du g&egrave;ne mpk1 pourrait &ecirc;tre augment&eacute;e par diff&eacute;rents sucres y compris le xylose, le tr&eacute;halose, glucose ou saccharose. mpk1 mutants null g&eacute;n&eacute;r&eacute;es par recombinaison homologue a grandi semblables &agrave; la souche sauvage de M. anisopliae sur milieu modifi&eacute; avec du xylose comme seule source de carbone. Bioessais insecte (Sphinx du tabac, Manduca sexta) indiquent cependant virulence significativement r&eacute;duite en Deltampk1. Les r&eacute;sultats de cette &eacute;tude sugg&egrave;rent que le Mpk1 horizontalement transf&eacute;r&eacute;s &agrave; M. anisopliae joue un r&ocirc;le d&#39;adaptation important cr&eacute;neau pour fongique propagation dans insecte h&eacute;moc&egrave;le. Apr&egrave;s le flux de glucides, de plantes aux insectes qui se nourrissent de plantes et d&#39;insectes champignons pathog&egrave;nes, est discute d&#39;une relation tritrophiques en liaison avec l&#39;exigence du parcours phosphoketolase fongiques.

Un phosphoketolase Mpk1 d&#39;origine bact&eacute;rienne est adaptable requis pour toute virulence chez l&#39;insect pathog&egrave;ne champignon Metarhizium anisopliae.

Metabolismo del pentosio attraverso il pathway phosphoketolase &egrave; stato ben caratterizzato nei batteri. In questa carta, segnaliamo l&#39;identificazione di un omologo di phosphoketolase Mpk1 nell&#39;insetto-patogeno fungo Metarhizium anisopliae. Analisi filogenetica ha mostrato che phosphoketolases fungine sono di origine batterica e si separarono in due superfamiglie. Gene frequente perdita o mancanza di acquisizione &egrave; evidente nelle specifiche lignaggi fungine o specie. Il gene mpk1 &egrave; altamente espresso Quando coltivate in ricchi di trealosio emolinfa degli insetti, ma scarsamente indotta dalla cuticola dell&#39;insetto o essudato radice vegetali ricchi di carboidrati. Inoltre, mpk1 gene expression ed enzima attivit&agrave; potrebbe essere sovraregolati da zuccheri differenti compreso xilosio, trealosio, glucosio o saccarosio. mpk1 null mutanti generati da ricombinazione omologa &egrave; cresciuta simile al wild type di M. anisopliae sul mezzo modificato con xilosio come un&#39;unica fonte di carbonio. Tuttavia, le analisi biologiche insetto (tobacco hornworm, Manduca sexta) ha mostrato significativamente ridotta virulenza in Deltampk1. I risultati di questo studio suggeriscono che il Mpk1 trasferito orizzontalmente in M. anisopliae svolge un ruolo di adattamento importante nicchia per la propagazione di funghi in haemocoel dell&#39;insetto. In seguito il flusso dei carboidrati dalle piante per alimentazione impianto di insetti e funghi patogeni degli insetti, un rapporto tritrofiche &egrave; discusso in associazione con il requisito di pathway phosphoketolase fungine.

Un phosphoketolase Mpk1 di origine batterica &egrave; adattivo richiesto per piena virulenza in insetto-patogeno fungo Metarhizium anisopliae.

Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ~30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ~16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogeneous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains with customized properties.

Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum.

Metarhizium spp sont utilis&eacute;s comme des alternatives &eacute;cologiques aux insecticides chimiques, comme syst&egrave;mes mod&egrave;les pour &eacute;tudie les interactions insecte-champignon et comme une ressource de g&egrave;nes pour la biotechnologie. Nous pr&eacute;sentons une analyse comparative des s&eacute;quences du g&eacute;nome du large spectre insecte pathog&egrave;ne Metarhizium anisopliae et le m&ecirc;me sp&eacute;cifiques M. acridum. Les analyses du g&eacute;nome entier indiquent que les structures du g&eacute;nome de ces deux esp&egrave;ces sont hautement synt&eacute;niques et que le genre Metarhizium a &eacute;volu&eacute; des endophytes de plante ou d&#39;agents pathog&egrave;nes. M. anisopliae tant M. acridum ont une tr&egrave;s grande proportion de g&egrave;nes codant des prot&eacute;ines s&eacute;cr&eacute;t&eacute;es que les autres champignons, alors qu&#39;environ 30 % d&#39;entre eux n&#39;ont pas fonctionnellement caract&eacute;ris&eacute;s homologues, sugg&eacute;rant jusqu&#39;alors insoup&ccedil;onn&eacute;es d&#39;interactions entre les pathog&egrave;nes fongiques et d&#39;insectes. L&#39;analyse des g&egrave;nes de la transposase donn&eacute; preuve de r&eacute;p&eacute;t&eacute;es par des mutations ponctuelles qui se produisent chez M. acridum mais non chez M. anisopliae. Avec l&#39;aide de la base de g&egrave;ne interaction h&ocirc;te-pathog&egrave;ne, ~ 16 % de Metarhizium g&egrave;nes ont &eacute;t&eacute; identifi&eacute;s qui sont semblables aux exp&eacute;rimentalement v&eacute;rifi&eacute;es de g&egrave;nes impliqu&eacute;s dans la pathog&eacute;nie chez d&#39;autres champignons, en particulier les agents pathog&egrave;nes des plantes. Toutefois, par rapport &agrave; M. acridum, M. anisopliae a &eacute;volu&eacute; avec plusieurs familles de g&egrave;ne &eacute;largi des prot&eacute;ases, chitinases, cytochrome P450s, polyketide synthases et synth&eacute;tases peptides ribosomiques pour cuticules-d&eacute;gradation, d&eacute;sintoxication et biosynth&egrave;se de toxines qui peut-&ecirc;tre faciliter sa capacit&eacute; &agrave; s&#39;adapter &agrave; des environnements h&eacute;t&eacute;rog&egrave;nes. L&#39;analyse transcriptionnelle des deux champignons au cours du processus d&#39;infection pr&eacute;coce fourni davantage pistes sur les g&egrave;nes et les voies impliqu&eacute;es dans l&#39;infectiosit&eacute; et la sp&eacute;cificit&eacute;. Mention particuli&egrave;re, M. acridum transcrit distinctes r&eacute;cepteurs G-prot&eacute;ine a coupl&eacute; sur les cuticules de criquets (les h&ocirc;tes naturels) et les cafards, tandis que M. anisopliae transcrit le m&ecirc;me r&eacute;cepteur sur les deux h&ocirc;tes. Cette &eacute;tude facilitera l&#39;identification des g&egrave;nes de virulence et le d&eacute;veloppement de souches am&eacute;lior&eacute;es de lutte biologique avec des propri&eacute;t&eacute;s personnalis&eacute;es.

Le s&eacute;quen&ccedil;age du g&eacute;nome et transcriptomique comparative du mod&egrave;le champignons entomopathog&egrave;ne Metarhizium anisopliae et M. acridum.

Metarhizium spp vengono utilizzati come alternative ecocompatibili di insetticidi chimici, come sistemi modello per studiare le interazioni insetto-fungo e come una risorsa di geni per la biotecnologia. Vi presentiamo un&#39;analisi comparativa delle sequenze del genoma del patogeno ad ampio spettro d&#39;insetti Metarhizium anisopliae e la acridum di M. acridid-specifiche. Intero genoma analisi indicano che le strutture del genoma di queste due specie sono altamente syntenic e suggeriscono che il genere Metarhizium evoluto da pianta endofiti o agenti patogeni. Sia M. anisopliae e M. acridum hanno una percentuale sorprendentemente maggiore di geni che codificano per proteine secrete di altri funghi, mentre ~ 30% di questi hanno caratterizzato non funzionalmente omologhi, suggerendo finora insospettate interazioni tra funghi patogeni e insetti. L&#39;analisi dei geni ha fornito prove di ripetizione-indotto mutazioni puntiformi che si verificano in M. acridum, ma non in M. anisopliae. Con l&#39;aiuto del database di gene interazione ospite-patogeno, ~ 16% di Metarhizium geni sono stati identificati che sono simili a sperimentalmente verificato geni coinvolti nella patogenicit&agrave; in altri funghi, particolarmente agenti patogeni vegetali. Tuttavia, rispetto al M. acridum, M. anisopliae ha evoluto con molte famiglie di espanso gene della proteasi, chitinases, citocromo P450s, polyketide synthases e sintetasi del peptide nonribosomal per cuticola-degradazione, disintossicazione e biosintesi di tossina che pu&ograve; facilitare la sua capacit&agrave; di adattarsi ad ambienti eterogenei. Analisi trascrizionale di entrambi funghi durante i processi di infezione precoce fornito ulteriori intuizioni i geni e pathways coinvolti nella infettivit&agrave; e specificit&agrave;. Di particolare nota, M. acridum trascritto distinti recettori proteina G accoppiata su cuticole da locuste (i padroni di casa naturale) e scarafaggi, mentre M. anisopliae trascritto il recettore stesso su entrambi gli host. Questo studio facilitano l&#39;identificazione di geni di virulenza e lo sviluppo di ceppi di biocontrollo migliorata con propriet&agrave; personalizzate.

Il sequenziamento del genoma e trascrittomica comparativa del modello funghi entomopatogeni Metarhizium anisopliae e M. acridum.

Insetti funghi patogeni come Beauveria bassiana sono stati sviluppati come agenti di biocontrollo ecologico contro parassiti artropodi. Tuttavia, restrittive fattori ambientali, tra cui frequentemente solare radiazione ultravioletta (UV) portano a prestazioni campo incoerente. Per migliorare la resistenza ai danni UV, abbiamo utilizzato trasformazione mediata da Agrobacterium per ingegnere b. bassiana con un gene esogeno tirosinasi. I risultati hanno mostrato che il transformants mitoticamente stabile prodotto grandi quantit&agrave; di pigmenti giallastri rispetto al ceppo wild-type, e questi impartita significativamente aumentata resistenza ai raggi UV. La virulenza dell&#39;isolato transgenico &egrave; stato anche significativamente aumentata contro il baco da seta Bombyx mori e il piatto alimentare per vermi Tenebrio molitor. Questo studio ha dimostrato che l&#39;ingegneria genetica di b. bassiana con un gene tirosinasi &egrave; un modo efficace per migliorare la tolleranza fungina contro danni UV.

Migliorare la resistenza ai raggi UV e la virulenza di Beauveria bassiana di ingegneria genetica con un gene esogeno tirosinasi.

Insectes champignons pathog&egrave;nes comme Beauveria bassiana ont &eacute;t&eacute; &eacute;labor&eacute;s en tant qu&#39;agents de lutte biologique respectueux de l&#39;environnement contre les arthropodes nuisibles. Toutefois, les facteurs environnementaux restrictifs, notamment solaires ultraviolets (UV) fr&eacute;quemment entra&icirc;ner performance sur le terrain incompatible. Pour am&eacute;liorer la r&eacute;sistance aux rayons UV, nous avons utilis&eacute; la transformation par Agrobacterium &agrave; l&#39;ing&eacute;nieur b. bassiana avec un g&egrave;ne de la tyrosinase exog&egrave;ne. Les r&eacute;sultats ont montr&eacute; que les transformants stables &agrave; la mitose produit de plus grandes quantit&eacute;s de pigments jaun&acirc;tres que la souche sauvage et ces donnait nettement en hausse, r&eacute;sistance aux UV. La virulence de la souche transg&eacute;nique &eacute;tait aussi significativement augment&eacute;e contre le ver &agrave; soie Bombyx mori et le t&eacute;n&eacute;brion Tenebrio molitor. Cette &eacute;tude a d&eacute;montr&eacute; que le g&eacute;nie g&eacute;n&eacute;tique de b. bassiana avec un g&egrave;ne de la tyrosinase est un moyen efficace d&#39;am&eacute;liorer la tol&eacute;rance fongique contre les rayons UV.

Am&eacute;liorer la r&eacute;sistance aux UV et la virulence de Beauveria bassiana par g&eacute;nie g&eacute;n&eacute;tique avec un g&egrave;ne de la tyrosinase exog&egrave;ne.

Insect pathogenic fungi like Beauveria bassiana have been developed as environmentally friendly biocontrol agents against arthropod pests. However, restrictive environmental factors, including solar ultraviolet (UV) radiation frequently lead to inconsistent field performance. To improve resistance to UV damage, we used Agrobacterium-mediated transformation to engineer B. bassiana with an exogenous tyrosinase gene. The results showed that the mitotically stable transformants produced larger amounts of yellowish pigments than the wild-type strain, and these imparted significantly increased UV-resistance. The virulence of the transgenic isolate was also significantly increased against the silkworm Bombyx mori and the mealworm Tenebrio molitor. This study demonstrated that genetic engineering of B. bassiana with a tyrosinase gene is an effective way to improve fungal tolerance against UV damage.

Improving UV resistance and virulence of Beauveria bassiana by genetic engineering with an exogenous tyrosinase gene.

Voltage-gated K(+) channels of the Shaw family (also known as the KCNC or Kv3 family) play pivotal roles in mammalian brains, and genetic or pharmacological disruption of their activities in mice results in a spectrum of behavioral defects. We have used the model system of Caenorhabditis elegans to elucidate conserved molecular mechanisms that regulate these channels. We have now found that the C. elegans Shaw channel KHT-1, and its mammalian homologue, murine Kv3.1b, are both modulated by acid phosphatases. Thus, the C. elegans phosphatase ACP-2 is stably associated with KHT-1, while its mammalian homolog, prostatic acid phosphatase (PAP; also known as ACPP-201) stably associates with murine Kv3.1b K(+) channels in vitro and in vivo. In biochemical experiments both phosphatases were able to reverse phosphorylation of their associated channel. The effect of phosphorylation on both channels is to produce a decrease in current amplitude and electrophysiological analyses demonstrated that dephosphorylation reversed the effects of phosphorylation on the magnitude of the macroscopic currents. ACP-2 and KHT-1 were colocalized in the nervous system of C. elegans and, in the mouse nervous system, PAP and Kv3.1b were colocalized in subsets of neurons, including in the brain stem and the ventricular zone. Taken together, this body of evidence suggests that acid phosphatases are general regulatory partners of Shaw-like K(+) channels.

An evolutionarily conserved mode of modulation of Shaw-like Kâº channels.

Voltage-d&eacute;pendant des canaux k de la famille Shaw (&eacute;galement connu sous le nom la famille KCNC ou Kv3) jouent un r&ocirc;le essentiel dans les cerveaux de mammif&egrave;res et des perturbations g&eacute;n&eacute;tiques ou pharmacologiques de leurs activit&eacute;s dans la souris donne un spectre d&#39;anomalies comportementales. Nous avons utilis&eacute; le syst&egrave;me de mod&egrave;le de Caenorhabditis elegans pour &eacute;lucider les m&eacute;canismes mol&eacute;culaires conserv&eacute;s qui r&eacute;gissent ces canaux. Maintenant, nous avons trouv&eacute; que les c. elegans Shaw canal KHT-1, et son homologue mammalien, murin Kv3.1b, sont tous deux modul&eacute;es par les phosphatases acides. Ainsi, la phosphatase de c. elegans ACP-2 est associ&eacute; &agrave; stablement KHT-1, tandis que son homologue chez les mammif&egrave;res, la phosphatase acide prostatique (PAP ; &eacute;galement connu sous le nom ACPP-201) associe stablement murines canaux k Kv3.1b in vitro et in vivo. Au cours d&#39;exp&eacute;riences biochimiques les deux phosphatases ont pu inverser la phosphorylation de leur canal associ&eacute;. L&#39;effet de la phosphorylation sur deux canaux est de produire une diminution de l&#39;amplitude du courant et analyses &eacute;lectrophysiologiques ont montr&eacute; cette d&eacute;phosphorylation renvers&eacute; les effets de la phosphorylation sur l&#39;amplitude des courants macroscopiques. ACP-2 et KHT-1 ont &eacute;t&eacute; log&eacute;es dans le syst&egrave;me nerveux de c. elegans et, dans le syst&egrave;me nerveux de la souris, PAP et Kv3.1b ont &eacute;t&eacute; log&eacute;es dans des sous-ensembles des neurones, notamment dans le tronc c&eacute;r&eacute;bral et de la zone ventriculaire. Pris ensemble, cette accumulation de preuves sugg&egrave;re que les phosphatases acides sont commandit&eacute;s r&eacute;glementaires des canaux de Shaw comme k.

Un mode &eacute;volutif conserv&eacute; de la modulation des canaux K&#x207A; Shaw-comme.

Tensione-gated K(+) canali della famiglia Shaw (anche conosciuto come la famiglia KCNC o Kv3) svolgono un ruolo cruciale nel cervello dei mammiferi e disturbo genetico o farmacologico della loro attivit&agrave; nei topi si traduce in uno spettro di difetti comportamentali. Abbiamo usato il sistema di modello di Caenorhabditis elegans delucidare conservati meccanismi molecolari che regolano questi canali. Ora abbiamo trovato che il c. elegans Shaw canale KHT-1, e suo omologo mammifero, Kv3.1b murino, sono modulati da fosfatasi acide. Cos&igrave;, la fosfatasi di c. elegans ACP-2 &egrave; stabilmente associato KHT-1, mentre suo omologo mammifero, fosfatasi acida prostatica (PAP; anche conosciuto come ACPP-201) associa stabilmente con murini Kv3.1b K(+) canali in vitro e in vivo. In esperimenti biochimici sia fosfatasi sono stati in grado di invertire la fosforilazione del loro canale associato. L&#39;effetto di fosforilazione su entrambi i canali &egrave; quello di produrre una diminuzione di ampiezza corrente e analisi elettrofisiologiche dimostrato che defosforilazione invertita gli effetti della fosforilazione sulla grandezza delle correnti macroscopiche. ACP-2 e KHT-1 erano colocalized nel sistema nervoso di c. elegans e, nel sistema nervoso del mouse, PAP e Kv3.1b furono colocalized in sottoinsiemi di neuroni, comprese nel tronco cerebrale e la zona ventricolare. Presi insieme, questo corpo di prove suggerisce che la fosfatasi acide sono partner regolamentazione generale dei canali Shaw-come K(+).

Una modalit&agrave; evolutivamente conservata di modulazione dei canali Shaw-come K&#x207A;.

Autophagy is a highly conserved process that maintains intracellular homeostasis by degrading proteins or organelles in all eukaryotes. The effect of autophagy on fungal biology and infection of insect pathogens is unknown. Here, we report the function of MrATG8, an ortholog of yeast ATG8, in the entomopathogenic fungus Metarhizium robertsii. MrATG8 can complement an ATG8-defective yeast strain and deletion of MrATG8 impaired autophagy, conidiation and fungal infection biology in M. robertsii. Compared with the wild-type and gene-rescued mutant, Mratg8Î” is not inductive to form the infection-structure appressorium and is impaired in defense response against insect immunity. In addition, accumulation of lipid droplets (LDs) is significantly reduced in the conidia of Mratg8Î” and the pathogenicity of the mutant is drastically impaired. We also found that the cellular level of a LD-specific perilipin-like protein is significantly lowered by deletion of MrATG8 and that the carboxyl terminus beyond the predicted protease cleavage site is dispensable for MrAtg8 function. To corroborate the role of autophagy in fungal physiology, the homologous genes of yeast ATG1, ATG4 and ATG15, designated as MrATG1, MrATG4 and MrATG15, were also deleted in M. robertsii. In contrast to Mratg8Î”, these mutants could form appressoria, however, the LD accumulation and virulence were also considerably impaired in the mutant strains. Our data showed that autophagy is required in M. robertsii for fungal differentiation, lipid biogenesis and insect infection. The results advance our understanding of autophagic process in fungi and provide evidence to connect autophagy with lipid metabolism.

Linkage of autophagy to fungal development, lipid storage and virulence in Metarhizium robertsii.

Autophagie est un processus hautement conserv&eacute; qui maintient l&#39;hom&eacute;ostasie intracellulaire en d&eacute;gradant les prot&eacute;ines ou les organites chez tous les eucaryotes. L&#39;effet de l&#39;autophagie sur biologie fongique et infection des insectes pathog&egrave;nes sont inconnues. Nous rapportons ici, la fonction de MrATG8, un orthologue de levure ATG8, dans le robertsii de Metarhizium champignon entomopathog&egrave;ne. MrATG8 peut compl&eacute;ter une souche de levure ATG8-d&eacute;fectueux et suppression de MrATG8 alt&eacute;r&eacute;s autophagie, conidiation et biologie d&#39;infection fongique &agrave; M. robertsii. En comparaison avec le type sauvage et a sauv&eacute; le g&egrave;ne mutant, Mratg8&Delta; n&#39;est pas inductive pour former l&#39;appressorium infection-structure et est alt&eacute;r&eacute;e chez les r&eacute;action de d&eacute;fense contre les insecte immunit&eacute;. En outre, l&#39;accumulation de gouttelettes lipidiques (LDs) est significativement r&eacute;duite dans les conidies du Mratg8&Delta; et la pathog&eacute;nicit&eacute; du mutant est consid&eacute;rablement alt&eacute;r&eacute;e. Nous avons &eacute;galement constat&eacute; que le niveau cellulaire d&#39;une prot&eacute;ine sp&eacute;cifique LD perilipin est significativement abaiss&eacute; par la suppression de MrATG8 et que l&#39;extr&eacute;mit&eacute; carboxyle au-del&agrave; du site de clivage de prot&eacute;ase pr&eacute;dite est superflue pour la fonction MrAtg8. Pour corroborer le r&ocirc;le de l&#39;autophagie dans physiologie fongique, les g&egrave;nes homologues de levure ATG/1, ATG4 et ATG15, appel&eacute;s MrATG1, MrATG4 et MrATG15, ont &eacute;t&eacute; &eacute;galement supprim&eacute;s dans M. robertsii. Contrairement aux Mratg8&Delta;, ces mutants pourraient former des appressoria, cependant, l&#39;accumulation de LD et la virulence ont &eacute;t&eacute; &eacute;galement consid&eacute;rablement alt&eacute;r&eacute;es chez les souches mutantes. Nos r&eacute;sultats montrent que l&#39;autophagie est n&eacute;cessaire &agrave; M. robertsii pour la diff&eacute;renciation fongique, biogen&egrave;se des lipides et insecte infection. Les r&eacute;sultats faire progresser notre compr&eacute;hension des processus autophagique chez les champignons et fournissent des preuves pour connecter l&#39;autophagie avec le m&eacute;tabolisme des lipides.

Couplage de l&#39;autophagie d&eacute;veloppement fongique, stockage de lipides et virulence chez Metarhizium robertsii.

Autophagy &egrave; un processo altamente conservato che mantiene l&#39;omeostasi intracellulare degradando le proteine o organelli in tutti gli eucarioti. L&#39;effetto di autophagy su biologia fungosa e infezione di agenti patogeni dell&#39;insetto &egrave; sconosciuti. Riportiamo la funzione di MrATG8, un ortholog di lievito ATG8, nell&#39;entomopatogeni fungo Metarhizium robertsii. MrATG8 possono completare un ceppo di lievito ATG8 difettosi ed eliminazione di MrATG8 alterata autophagy, conidiation e biologia di infezione fungina in M. robertsii. Confrontato con il wild-type e salvato gene mutante, Mratg8&Delta; non &egrave; di tipo induttivo per formare la appressorium infezione-struttura ed &egrave; alterata in risposta di difesa contro l&#39;immunit&agrave; degli insetti. Inoltre, accumulo di gocce lipidiche (LDs) &egrave; significativamente ridotto i conidi di Mratg8&Delta; e la patogenicit&agrave; del mutante &egrave; drasticamente ridotta. Abbiamo anche trovato che il livello cellulare di una proteina simile perilipina LD-specifico &egrave; abbassato significativamente dall&#39;eliminazione di MrATG8 e che il terminale carbossilico oltre il sito di clivaggio proteasi previsto &egrave; superfluo per la funzione MrAtg8. Per corroborare il ruolo di autophagy nella fisiologia fungina, i geni omologhi del lievito ATG1, ATG4 e ATG15, indicato come MrATG1, MrATG4 e MrATG15, sono stati eliminati anche in M. robertsii. A differenza di Mratg8&Delta;, questi mutanti potrebbero formare appressoria, tuttavia, l&#39;accumulo di LD e virulenza furono anche notevolmente alterate in ceppi mutanti. I nostri dati hanno mostrato che autophagy &egrave; necessaria in M. robertsii per differenziazione fungine, biogenesi dei lipidi e infezione dell&#39;insetto. I risultati avanzano la nostra comprensione del processo autophagic in funghi e forniscano prove per collegare autophagy con metabolismo lipidico.

Zhibing Duan

Department of Neuroscience and Cell Biology,
Robert Wood Johnson Medical School,
Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School

University of Medicine and Dentistry of New Jersey

A Caenorhabditis elegans Model System for Amylopathy Study

Zhibing Duan

Department of Neuroscience and Cell Biology,Robert Wood Johnson Medical School,Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School

University of Medicine and Dentistry of New Jersey

A Caenorhabditis elegans Model System for Amylopathy Study

Department of Neuroscience and Cell Biology,
Robert Wood Johnson Medical School,
Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School