tandem-high-pressure-freezing-quick-freeze-substitution-plant-tissues

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

University of Tennessee, Knoxville

&#x75C5;&#x6BD2;&#x8BF1;&#x5BFC;&#x7684;&#x57FA;&#x56E0;&#x6C89;&#x9ED8; (VIGS) &#x662F;&#x6700;&#x8FD1;&#x5F00;&#x53D1;&#x7684;&#x57FA;&#x56E0;&#x8A8A;&#x672C;&#x5236;&#x6B62;&#x6280;&#x672F;&#x8868;&#x5F81;&#x7684;&#x690D;&#x7269;&#x57FA;&#x56E0;&#x529F;&#x80FD;&#x3002;&#x65B9;&#x6CD5;&#x5305;&#x62EC;&#x514B;&#x9686;&#x5230;&#x75C5;&#x6BD2;&#x8F7D;&#x4F53;&#x7684;&#x6709;&#x9488;&#x5BF9;&#x6027;&#x7684;&#x690D;&#x7269;&#x57FA;&#x56E0;&#x77ED;&#x5E8F;&#x5217;&#x3002;&#x4F7F;&#x7528;&#x5411;&#x91CF;&#x6765;&#x611F;&#x67D3;&#x4E86;&#x5E74;&#x8F7B;&#x7684;&#x5DE5;&#x5382;&#xFF0C;&#x5E76;&#x5728;&#x51E0;&#x4E2A;&#x661F;&#x671F;&#x65E8;&#x5728;&#x6291;&#x5236;&#x75C5;&#x6BD2;&#x590D;&#x5236;&#x7684;&#x690D;&#x7269;&#x7684;&#x81EA;&#x7136;&#x9632;&#x5FA1;&#x673A;&#x5236;&#x8FD8;&#x5BFC;&#x81F4;&#x5177;&#x4F53;&#x9000;&#x5316;&#x7684;&#x5206;&#x6CCC;&#x9488;&#x5BF9;&#x6C89;&#x9ED8;&#x7684;&#x5185;&#x6E90;&#x6027;&#x690D;&#x7269;&#x57FA;&#x56E0;&#x4ECE;&#x3002;VIGS &#x662F;&#x5FEB;&#x901F; &#xFF08;3-4 &#x5468;&#x4ECE;&#x611F;&#x67D3;&#x5230;&#x6C89;&#x9ED8;&#xFF09;&#xFF0C;&#x4E0D;&#x9700;&#x8981;&#x53D1;&#x5C55;&#x7A33;&#x5B9A;&#x690D;&#x682A;&#x3001; &#x5141;&#x8BB8;&#x7684;&#x8868;&#x578B;&#xFF0C;&#x53EF;&#x80FD;&#x5728;&#x7A33;&#x5B9A;&#x7684;&#x884C;&#x4E2D;&#xFF0C;&#x81F4;&#x547D;&#x6027;&#x548C;&#x6F5C;&#x529B;&#x6C89;&#x9ED8;&#x4E2A;&#x4EBA;&#x6216;&#x591A;&#x4E2A;&#x57FA;&#x56E0;&#x5BB6;&#x65CF;&#x6210;&#x5458;&#x7684;&#x8868;&#x5F81;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x7B80;&#x8981;&#x56DE;&#x987E;&#x4E00;&#x4E0B;&#x53D1;&#x73B0;&#x5BFC;&#x81F4; VIGS &#x548C;&#x6240;&#x8C13;&#x7684;&#x6709;&#x5173;&#x5B9E;&#x9A8C;&#x7684;&#x8981;&#x6C42;&#x7684;&#x6709;&#x6548;&#x7684;&#x6C89;&#x9ED8;&#x7684;&#x53D1;&#x5C55;&#x3002;&#x6211;&#x4EEC;&#x63CF;&#x8FF0; VIGS &#x548C;&#x5982;&#x4F55;&#x53EF;&#x4EE5;&#x4F18;&#x5316;&#x548C;&#x7528;&#x4E8E;&#x8FD9;&#x4E24;&#x4E2A;&#x6B63;&#x5411;&#x548C;&#x53CD;&#x5411;&#x9057;&#x4F20;&#x5B66;&#x7814;&#x7A76;&#x7684;&#x65B9;&#x6CD5;&#x3002;&#x4EE5;&#x53CA;&#x5982;&#x4F55;&#x53EF;&#x80FD;&#x514B;&#x670D;&#x7684;&#x5C40;&#x9650;&#x6027; VIGS &#x8BA8;&#x8BBA; VIGS &#x76F8;&#x6BD4;&#x5176;&#x4ED6;&#x529F;&#x80FD;&#x635F;&#x5931;&#x7684;&#x529E;&#x6CD5;&#x4F9B;&#x690D;&#x7269;&#x7684;&#x5229;&#x4E0E;&#x5F0A;&#x3002;&#x4F8B;&#x5B50;&#x5BA1;&#x67E5; VIGS &#x88AB;&#x7528;&#x6765;&#x63D0;&#x4F9B;&#x53D1;&#x5C55;&#x548C;&#x690D;&#x7269;&#x9632;&#x5FA1;&#x53CD;&#x5E94;&#x7684;&#x7279;&#x5B9A;&#x57FA;&#x56E0;&#x5728;&#x690D;&#x7269;&#x4E2D;&#x7684;&#x89D2;&#x8272;&#x91CD;&#x8981;&#x7684;&#x65B0;&#x89C1;&#x89E3;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x68C0;&#x67E5;&#x7684;&#x672A;&#x6765;&#x524D;&#x666F;&#x7684; VIGS &#x4F5C;&#x4E3A;&#x8BC4;&#x4F30;&#x548C;&#x5B9A;&#x6027;&#x7684;&#x690D;&#x7269;&#x57FA;&#x56E0;&#x529F;&#x80FD;&#x7684;&#x5F3A;&#x5927;&#x5DE5;&#x5177;&#x3002;

&#x5E94;&#x7528;&#x7A0B;&#x5E8F;&#x548C;&#x75C5;&#x6BD2;&#x8BF1;&#x5BFC;&#x7684;&#x57FA;&#x56E0;&#x6C89;&#x9ED8;&#x7684;&#x690D;&#x7269;&#x57FA;&#x56E0;&#x529F;&#x80FD;&#x7684;&#x7814;&#x7A76;&#x4F18;&#x52BF;&#x3002;

Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. The approach involves cloning a short sequence of a targeted plant gene into a viral delivery vector. The vector is used to infect a young plant, and in a few weeks natural defense mechanisms of the plant directed at suppressing virus replication also result in specific degradation of mRNAs from the endogenous plant gene that is targeted for silencing. VIGS is rapid (3-4 weeks from infection to silencing), does not require development of stable transformants, allows characterization of phenotypes that might be lethal in stable lines, and offers the potential to silence either individual or multiple members of a gene family. Here we briefly review the discoveries that led to the development of VIGS and what is known about the experimental requirements for effective silencing. We describe the methodology of VIGS and how it can be optimized and used for both forward and reverse genetics studies. Advantages and disadvantages of VIGS compared with other loss-of-function approaches available for plants are discussed, along with how the limitations of VIGS might be overcome. Examples are reviewed where VIGS has been used to provide important new insights into the roles of specific genes in plant development and plant defense responses. Finally, we examine the future prospects for VIGS as a powerful tool for assessing and characterizing the function of plant genes.

Applications and advantages of virus-induced gene silencing for gene function studies in plants.

Induite par le virus gene silencing (VIGS) est une technique d&#xE9;velopp&#xE9;e r&#xE9;cemment g&#xE8;ne transcription de suppression pour caract&#xE9;riser la fonction des g&#xE8;nes de plantes. L&#x26;#39;approche implique le clonage d&#x26;#39;une courte s&#xE9;quence d&#x26;#39;un g&#xE8;ne v&#xE9;g&#xE9;tal cibl&#xE9; dans un vecteur viral de livraison. Le vecteur est utilis&#xE9; pour infecter une jeune plante, et en quelques semaines des m&#xE9;canismes de d&#xE9;fense naturels de la plante dirig&#xE9; &#xE0; la r&#xE9;plication du virus supprimant &#xE9;galement entra&#xEE;ner une d&#xE9;gradation sp&#xE9;cifique des ARNm du g&#xE8;ne endog&#xE8;ne de plante qui est cibl&#xE9; pour faire taire. VIGS est rapide (3-4 semaines de l&#x26;#39;infection &#xE0; silencing), ne n&#xE9;cessite pas de d&#xE9;veloppement de transformants stables, permet la caract&#xE9;risation de ph&#xE9;notypes qui pourraient &#xEA;tre mortelle chez les lign&#xE9;es stables, et offre la possibilit&#xE9; de garder le silence soit des membres individuels ou multiples d&#x26;#39;une famille de g&#xE8;nes. Ici, nous bri&#xE8;vement en revue les d&#xE9;couvertes qui ont men&#xE9; &#xE0; l&#x26;#39;&#xE9;laboration de VIGS et ce qui est connu au sujet des exigences exp&#xE9;rimentales pour faire taire efficace. Nous d&#xE9;crivons la m&#xE9;thodologie de VIGS et comment elle peut &#xEA;tre optimis&#xE9;e et utilis&#xE9;e pour les &#xE9;tudes de g&#xE9;n&#xE9;tique &#xE0; la fois avant et arri&#xE8;re. Avantages et inconv&#xE9;nients de VIGS par rapport aux autres la perte de fonction des approches disponibles pour les plantes sont discut&#xE9;s, ainsi que la fa&#xE7;on dont les limitations de VIGS pourraient &#xEA;tre surmont&#xE9;s. Des exemples sont examin&#xE9;s o&#xF9; VIGS a &#xE9;t&#xE9; utilis&#xE9; pour fournir des indications importantes sur le r&#xF4;le des g&#xE8;nes sp&#xE9;cifiques du d&#xE9;veloppement des plantes et des r&#xE9;ponses de d&#xE9;fense des plantes. Enfin, nous examinons les perspectives d&#x26;#39;avenir pour VIGS comme un outil puissant pour &#xE9;valuer et caract&#xE9;riser la fonction des g&#xE8;nes de plantes.

Applications et avantages de virus induite par Gene Silencing pour les &#xE9;tudes fonction des g&#xE8;nes dans les plantes

Anwendungsm&#xF6;glichkeiten und Vorteile der Virus-induzierte Gen-Silencing f&#xFC;r die Gen-Funktions-Studien in Pflanzen.

Silenziamento genico indotto da virus (VIGS) &egrave; una tecnica di soppressione di trascrizione genica recentemente sviluppati per caratterizzare la funzione dei geni della pianta. L&#39;approccio prevede una breve sequenza di un gene di pianta mirati in un vettore virale consegna la clonazione. Il vettore &egrave; usato per infettare una pianta giovane, e in poche settimane dei meccanismi naturali di difesa della pianta diretto a sopprimere la replicazione del virus anche tradursi in degrado specifico dei mRNAs dal gene endogeno pianta che &egrave; mirato per tacere. VIGS &egrave; rapida (3-4 settimane dall&#39;infezione di silenziamento), non richiede lo sviluppo di transformants stabile, consente la caratterizzazione fenotipica che potrebbe essere letale in linee stabili e offre il potenziale per mettere a tacere o singoli o pi&ugrave; membri di una famiglia di geni. Qui rivediamo brevemente le scoperte che hanno portato allo sviluppo di VIGS e ci&ograve; che &egrave; noto circa i requisiti sperimentali per la tacitazione efficace. Descriviamo la metodologia di VIGS e come pu&ograve; essere ottimizzato e usato per entrambi gli studi di genetica di avanti e indietro. Vantaggi e svantaggi di VIGS rispetto ad altri approcci di perdita di funzione disponibile per le piante sono esposte, insieme a come potrebbero superare le limitazioni di VIGS. Esempi sono rivisti dove VIGS &egrave; stato utilizzato per fornire importanti nuove intuizioni i ruoli di specifici geni nello stabilimento di sviluppo e impianto di risposte di difesa. Infine, si esaminano le prospettive future per VIGS come un potente strumento per valutare e caratterizzare la funzione dei geni della pianta.

Applicazioni e vantaggi di silenziamento genico indotto da virus per gli studi di funzione genica in piante.

&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x8A98;&#x767A;&#x6027;&#x907A;&#x4F1D;&#x5B50;&#x30B5;&#x30A4;&#x30EC;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#xFF08;VIGS&#xFF09;&#x306F;&#x3001;&#x690D;&#x7269;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x7279;&#x5FB4;&#x3065;&#x3051;&#x308B;&#x305F;&#x3081;&#x306B;&#x6700;&#x8FD1;&#x958B;&#x767A;&#x3055;&#x308C;&#x305F;&#x907A;&#x4F1D;&#x5B50;&#x8EE2;&#x5199;&#x6291;&#x5236;&#x624B;&#x6CD5;&#x3067;&#x3042;&#x308B;&#x3002;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x306F;&#x3001;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x9001;&#x9054;&#x30D9;&#x30AF;&#x30BF;&#x30FC;&#x306B;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3092;&#x7D5E;&#x3063;&#x305F;&#x690D;&#x7269;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x30AF;&#x30ED;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x77ED;&#x3044;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x304C;&#x542B;&#x307E;&#x308C;&#x307E;&#x3059;&#x3002;&#x30D9;&#x30AF;&#x30C8;&#x30EB;&#x306F;&#x3001;&#x82E5;&#x3044;&#x690D;&#x7269;&#x3092;&#x611F;&#x67D3;&#x3055;&#x305B;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3001;&#x6570;&#x9031;&#x9593;&#x4EE5;&#x5185;&#x306B;&#x6291;&#x5236;&#x3059;&#x308B;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x8907;&#x88FD;&#x306B;&#x5411;&#x3051;&#x305F;&#x690D;&#x7269;&#x306E;&#x81EA;&#x7136;&#x306A;&#x9632;&#x5FA1;&#x6A5F;&#x69CB;&#x306F;&#x307E;&#x305F;&#x3001;&#x30B5;&#x30A4;&#x30EC;&#x30F3;&#x3092;&#x5BFE;&#x8C61;&#x3068;&#x3057;&#x3066;&#x3044;&#x308B;&#x5185;&#x56E0;&#x6027;&#x690D;&#x7269;&#x907A;&#x4F1D;&#x5B50;&#x304B;&#x3089;&#x306E;mRNA&#x306E;&#x7279;&#x7570;&#x7684;&#x5206;&#x89E3;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; VIGS&#x306F;&#x6025;&#x901F;&#x3067;&#x3042;&#x308B;&#xFF08;&#x611F;&#x67D3;&#x304B;&#x3089;&#x30B5;&#x30A4;&#x30EC;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x306B;3-4&#x9031;&#x9593;&#xFF09;&#x3001;&#x5B89;&#x5B9A;&#x3057;&#x305F;&#x5F62;&#x8CEA;&#x8EE2;&#x63DB;&#x4F53;&#x306E;&#x958B;&#x767A;&#x3092;&#x5FC5;&#x8981;&#x3068;&#x3057;&#x306A;&#x3044;&#x3001;&#x5B89;&#x5B9A;&#x3057;&#x305F;&#x30E9;&#x30A4;&#x30F3;&#x3067;&#x81F4;&#x6B7B;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x8868;&#x73FE;&#x578B;&#x306E;&#x7279;&#x6027;&#x8A55;&#x4FA1;&#x3092;&#x53EF;&#x80FD;&#x306B;&#x3057;&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x306E;&#x500B;&#x3005;&#x306E;&#x307E;&#x305F;&#x306F;&#x8907;&#x6570;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x306E;&#x3044;&#x305A;&#x308C;&#x304B;&#x3092;&#x9ED9;&#x3089;&#x305B;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x7C21;&#x5358;&#x306B;VIGS&#x3069;&#x306E;&#x3088;&#x3046;&#x306A;&#x52B9;&#x679C;&#x7684;&#x306A;&#x30B5;&#x30A4;&#x30EC;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x5B9F;&#x9A13;&#x306E;&#x8981;&#x4EF6;&#x306B;&#x3064;&#x3044;&#x3066;&#x306F;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x306E;&#x958B;&#x767A;&#x306B;&#x3064;&#x306A;&#x304C;&#x3063;&#x305F;&#x306E;&#x767A;&#x898B;&#x3092;&#x78BA;&#x8A8D;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005;&#x306F;VIGS&#x306E;&#x65B9;&#x6CD5;&#x8AD6;&#x3092;&#x8A18;&#x8FF0;&#x3059;&#x308B;&#x65B9;&#x6CD5;&#x3068;&#x3001;&#x305D;&#x308C;&#x304C;&#x6700;&#x9069;&#x5316;&#x3055;&#x308C;&#x3001;&#x9806;&#x65B9;&#x5411;&#x304A;&#x3088;&#x3073;&#x9006;&#x65B9;&#x5411;&#x306E;&#x4E21;&#x65B9;&#x306E;&#x907A;&#x4F1D;&#x5B66;&#x7814;&#x7A76;&#x306E;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x690D;&#x7269;&#x306E;&#x5229;&#x7528;&#x53EF;&#x80FD;&#x306A;&#x4ED6;&#x306E;&#x6A5F;&#x80FD;&#x55AA;&#x5931;&#x306E;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;VIGS&#x306E;&#x9577;&#x6240;&#x3068;&#x77ED;&#x6240;&#x306F;VIGS&#x306E;&#x9650;&#x754C;&#x304C;&#x514B;&#x670D;&#x3055;&#x308C;&#x308B;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x304B;&#x3068;&#x4E00;&#x7DD2;&#x306B;&#x3001;&#x8AAC;&#x660E;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002; VIGS&#x306F;&#x3001;&#x690D;&#x7269;&#x306E;&#x958B;&#x767A;&#x304A;&#x3088;&#x3073;&#x690D;&#x7269;&#x9632;&#x5FA1;&#x5FDC;&#x7B54;&#x306B;&#x304A;&#x3051;&#x308B;&#x7279;&#x5B9A;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x5F79;&#x5272;&#x306B;&#x91CD;&#x8981;&#x306A;&#x65B0;&#x3057;&#x3044;&#x6D1E;&#x5BDF;&#x3092;&#x63D0;&#x200B;&#x200B;&#x4F9B;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x5834;&#x6240;&#x306E;&#x4F8B;&#x304C;&#x898B;&#x76F4;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;&#x690D;&#x7269;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x8A55;&#x4FA1;&#x3057;&#x3001;&#x7279;&#x5FB4;&#x3065;&#x3051;&#x308B;&#x305F;&#x3081;&#x306E;&#x5F37;&#x529B;&#x306A;&#x30C4;&#x30FC;&#x30EB;&#x3068;&#x3057;&#x3066;VIGS&#x306E;&#x305F;&#x3081;&#x306B;&#x5C06;&#x6765;&#x306E;&#x898B;&#x901A;&#x3057;&#x3092;&#x8ABF;&#x3079;&#x307E;&#x3059;&#x3002;

&#x690D;&#x7269;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x6A5F;&#x80FD;&#x7814;&#x7A76;&#x306E;&#x305F;&#x3081;&#x306E;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x304A;&#x3088;&#x3073;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x8A98;&#x767A;&#x6027;&#x907A;&#x4F1D;&#x5B50;&#x30B5;&#x30A4;&#x30EC;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x306E;&#x5229;&#x70B9;

&#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC720;&#xB3C4; &#xC720;&#xC804;&#xC790; &#xCE68;&#xBB35; (VIGS) &#xC2DD;&#xBB3C; &#xC720;&#xC804;&#xC790;&#xC758; &#xAE30;&#xB2A5;&#xC744; &#xD2B9;&#xC131;&#xD654;&#xB97C; &#xC704;&#xD55C; &#xCD5C;&#xADFC;&#xC5D0; &#xAC1C;&#xBC1C;&#xD55C; &#xC720;&#xC804;&#xC790; &#xC0AC;&#xBCF8;&#xC744; &#xC5B5;&#xC81C; &#xAE30;&#xC220; &#xC774;&#xB2E4;. &#xBC29;&#xBC95;&#xC740; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC131; &#xC804;&#xB2EC; &#xBCA1;&#xD130;&#xC5D0; &#xB300;&#xC0C1;&#xB41C; &#xC2DD;&#xBB3C; &#xC720;&#xC804;&#xC790;&#xC758; &#xC9E7;&#xC740; &#xC2DC;&#xD000;&#xC2A4;&#xB97C; &#xBCF5;&#xC81C; &#xD569;&#xB2C8;&#xB2E4;. &#xBCA1;&#xD130;&#xB294; &#xC5B4;&#xB9B0; &#xC2DD;&#xBB3C;&#xC744; &#xAC10;&#xC5FC; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9; &#xD558; &#xACE0; &#xBA87; &#xC8FC; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xBCF5;&#xC81C; &#xC5B5;&#xC81C;&#xC5D0; &#xC9C0;&#xC2DC; &#xD558;&#xB294; &#xC2DD;&#xBB3C;&#xC758; &#xC790;&#xC5F0; &#xBC29;&#xC5B4; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xB610;&#xD55C; &#xC785;&#xC744; &#xC704;&#xD55C; &#xD0C0;&#xAC9F; &#xC0DD; &#xC2DD;&#xBB3C; &#xC720;&#xC804;&#xC790;&#xC5D0;&#xC11C; Mrnas&#xC758; &#xD2B9;&#xC815; &#xC800;&#xD558;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD;. VIGS&#xC740; &#xAE09;&#xC99D; (3-4 &#xC8FC; &#xAC10;&#xC5FC;&#xC5D0;&#xC11C; &#xC785;&#xC744;), &#xC548;&#xC815;&#xC801;&#xC778; transformants&#xC758; &#xAC1C;&#xBC1C;&#xC744; &#xD544;&#xC694;&#xB85C; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;, &#xC548;&#xC815;&#xC801;&#xC778; &#xB77C;&#xC778;&#xC5D0; &#xCE58;&#xBA85;&#xC801;&#xC77C; &#xC218;&#xB3C4; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAC1C;&#xC778; &#xB610;&#xB294; &#xC720;&#xC804;&#xC790; &#xAC00;&#xC871;&#xC758; &#xC5EC;&#xB7EC; &#xAD6C;&#xC131;&#xC6D0; &#xCE68;&#xBB35; &#xC7A0;&#xC7AC;&#xB825;&#xC744; &#xC81C;&#xACF5; &#xD558; &#xACE0;&#xAE30; &#xD2B9;&#xC131;&#xC744; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; VIGS &#xBC0F; &#xC54C;&#xB824;&#xC9C4; &#xD6A8;&#xACFC;&#xC801;&#xC778; &#xC785;&#xC744; &#xC704;&#xD55C; &#xC2E4;&#xD5D8; &#xC694;&#xAD6C; &#xC0AC;&#xD56D;&#xC5D0; &#xB300; &#xD55C; &#xAC1C;&#xBC1C;&#xC744; &#xBC1C;&#xACAC; &#xAC80;&#xD1A0;. &#xC6B0;&#xB9AC; VIGS &#xACE0; &#xC5B4;&#xB5BB;&#xAC8C; &#xCD5C;&#xC801;&#xD654; &#xB420; &#xC218; &#xC788;&#xB2E4; &#xACE0; &#xB450; &#xC815;&#xBC29;&#xD5A5; &#xBC0F; &#xC5ED;&#xBC29;&#xD5A5; &#xC720;&#xC804;&#xD559; &#xC5F0;&#xAD6C;&#xC5D0; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xBC29;&#xC2DD;&#xC5D0; &#xC124;&#xBA85;. &#xC5B4;&#xB5BB;&#xAC8C; VIGS&#xC758; &#xD55C;&#xACC4;&#xB97C; &#xADF9;&#xBCF5;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xD568;&#xAED8; &#xC2DD;&#xBB3C;&#xC5D0; &#xB300; &#xD55C; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xB2E4;&#xB978; &#xAE30;&#xB2A5; &#xC190;&#xC2E4; &#xBC29;&#xBC95;&#xC5D0; &#xBE44;&#xD574; VIGS&#xC758; &#xC7A5;&#xC810;&#xACFC; &#xB2E8;&#xC810; &#xC124;&#xBA85; &#xB418;&#xC5B4; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC608; &#xAC80;&#xD1A0; VIGS &#xAC1C;&#xBC1C; &#xBC0F; &#xC2DD;&#xBB3C; &#xBC29;&#xC5B4; &#xC751;&#xB2F5; &#xACF5;&#xC7A5;&#xC5D0;&#xC11C; &#xD2B9;&#xC815; &#xC720;&#xC804;&#xC790;&#xC758; &#xC5ED;&#xD560;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xD1B5;&#xCC30;&#xB825;&#xC744; &#xC81C;&#xACF5;&#xC5D0; &#xC0AC;&#xC6A9; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC6B0;&#xB9AC;&#xB97C; &#xD3C9;&#xAC00; &#xD558; &#xACE0; &#xC2DD;&#xBB3C; &#xC720;&#xC804;&#xC790;&#xC758; &#xAE30;&#xB2A5;&#xC744; &#xD2B9;&#xC131;&#xD654;&#xB97C; &#xC704;&#xD55C; &#xAC15;&#xB825;&#xD55C; &#xB3C4;&#xAD6C;&#xB85C; VIGS&#xC5D0; &#xB300; &#xD55C; &#xBBF8;&#xB798; &#xC804;&#xB9DD; &#xAC80;&#xD1A0;.

&#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8; &#xBC0F; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC720;&#xB3C4; &#xC720;&#xC804;&#xC790; &#xC2DD;&#xBB3C;&#xC5D0; &#xC720;&#xC804;&#xC790; &#xAE30;&#xB2A5; &#xC5F0;&#xAD6C;&#xC5D0; &#xB300; &#xD55C; &#xCE68;&#xBB35;&#xC758; &#xC7A5;&#xC810;.

Inducida por el virus de silenciamiento g&#xE9;nico (VIGS) es una transcripci&#xF3;n de genes recientemente desarrollada t&#xE9;cnica de supresi&#xF3;n para la caracterizaci&#xF3;n de la funci&#xF3;n de los genes de las plantas. El m&#xE9;todo implica la clonaci&#xF3;n una corta secuencia de un gen vegetal espec&#xED;fico en un vector de entrega viral. El vector se utiliza para infectar a una planta joven, y en pocas semanas los mecanismos naturales de defensa de la planta dirigida a la supresi&#xF3;n de la replicaci&#xF3;n del virus tambi&#xE9;n dan lugar a la degradaci&#xF3;n espec&#xED;fica de ARNm del gen vegetal end&#xF3;geno que est&#xE1; destinada a silenciar. VIGS es r&#xE1;pida (3-4 semanas de la infecci&#xF3;n a silenciar), no requiere el desarrollo de los transformantes estables, permite la caracterizaci&#xF3;n de fenotipos que pueden ser letales en l&#xED;neas estables, y ofrece la posibilidad de silenciar a cualquiera de los miembros individuales o m&#xFA;ltiples de una familia de genes. Aqu&#xED; un breve repaso de los descubrimientos que condujeron al desarrollo de VIGS y lo que se sabe acerca de los requerimientos experimentales para silenciar eficaz. Se describe la metodolog&#xED;a de VIGS y c&#xF3;mo puede ser optimizado y utilizado para estudios de gen&#xE9;tica directa e inversa. Ventajas y desventajas de VIGS comparado con otros p&#xE9;rdida de funci&#xF3;n de enfoques disponible para las plantas se tratan, junto con la forma de las limitaciones de VIGS podr&#xED;a ser superado. Ejemplos son revisados &#x200B;&#x200B;en VIGS ha sido utilizado para proporcionar nuevas e importantes ideas en el papel de genes espec&#xED;ficos en el desarrollo vegetal y las respuestas de defensa de la planta. Por &#xFA;ltimo, se examinan las perspectivas futuras de VIGS como una poderosa herramienta para evaluar y caracterizar la funci&#xF3;n de los genes de las plantas.

Aplicaciones y ventajas de los virus de silenciamiento g&#xE9;nico inducido por los estudios de funci&#xF3;n de genes en plantas

Plants have evolved in an environment rich with microorganisms that are eager to capitalize on the plants' biosynthetic and energy-producing capabilities. There are approximately 450 species of plant-pathogenic viruses, which cause a range of diseases. However, plants have not been passive in the face of these assaults, but have developed elaborate and effective defence mechanisms to prevent, or limit, damage owing to viral infection. Plant resistance genes confer resistance to various pathogens, including viruses. The defence response that is initiated after detection of a specific virus is stereotypical, and the cellular and physiological features associated with it have been well characterized. Recently, RNA silencing has gained prominence as an important cellular pathway for defence against foreign nucleic acids, including viruses. These pathways function in concert to result in effective protection against virus infection in plants.

Mechanisms of plant resistance to viruses.

Virus-induced gene silencing (VIGS) is a plant RNA-silencing technique that uses viral vectors carrying a fragment of a gene of interest to generate double-stranded RNA, which initiates the silencing of the target gene. Several viral vectors have been developed for VIGS and they have been successfully used in reverse genetics studies of a variety of processes occurring in plants. This approach has not been widely adopted for the model dicotyledonous species Arabidopsis (Arabidopsis thaliana), possibly because, until now, there has been no easy protocol for effective VIGS in this species. Here, we show that a widely used tobacco rattle virus-based VIGS vector can be used for silencing genes in Arabidopsis ecotype Columbia-0. The protocol involves agroinfiltration of VIGS vectors carrying fragments of genes of interest into seedlings at the two- to three-leaf stage and requires minimal modification of existing protocols for VIGS with tobacco rattle virus vectors in other species like Nicotiana benthamiana and tomato (Lycopersicon esculentum). The method described here gives efficient silencing in Arabidopsis ecotype Columbia-0. We show that VIGS can be used to silence genes involved in general metabolism and defense and it is also effective at knocking down expression of highly expressed transgenes. A marker system to monitor the progress and efficiency of VIGS is also described.

Efficient virus-induced gene silencing in Arabidopsis.

Plant innate immunity is mediated by Resistance (R) proteins, which bear a striking resemblance to animal molecules of similar function. Tobacco N is a TIR-NB-LRR R gene that confers resistance to Tobacco mosaic virus, specifically the p50 helicase domain. An intriguing question is how plant R proteins recognize the presence of pathogen-derived Avirulence (Avr) elicitor proteins. We have used biochemical cell fraction and immunoprecipitation in addition to confocal fluorescence microscopy of living tissue to examine the association between N and p50. Surprisingly, both N and p50 are cytoplasmic and nuclear proteins, and N's nuclear localization is required for its function. We also demonstrate an in planta association between N and p50. Further, we show that N's TIR domain is critical for this association, and indeed, it alone can associate with p50. Our results differ from current models for plant innate immunity that propose detection is mediated solely through the LRR domains of these molecules. The data we present support an intricate process of pathogen elicitor recognition by R proteins involving multiple subcellular compartments and the formation of multiple protein complexes.

A novel role for the TIR domain in association with pathogen-derived elicitors.

The Toll-interleukin-1 receptor (TIR) domain is found in innate immune molecules of insects, animals, and plants. There is a marked conservation of the signaling pathways used by TIR domain-containing immune receptors in animals. In plants, pathways involving TIR domain-containing proteins are not as well understood, but data are beginning to suggest important differences between the functions of plant and animal proteins. Plant Resistance proteins use their TIR domains for pathogen detection, and they may also function in the nucleus to direct the expression of genes involved in defense responses. This shows that plants have used their TIR domains to perform a multitude of duties and have expanded the functions of the TIR domain beyond those seen in animals.

The functions of plant TIR domains.

Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics. VIGS vectors derived from the Tobacco rattle virus (TRV) are among the most popular for VIGS. We have developed a TRV RNA2 vector that allows the insertion of gene silencing fragments by ligation-independent cloning (LIC). This new vector has several advantages over previous vectors, particularly for applications involving the analysis of large numbers of sequences, since TRV-LIC vectors containing the desired insert are obtained with 100% efficiency. Importantly, this vector allows the high-throughput cloning of silencing fragments without the use of costly enzymes required for recombination, as is the case with GATEWAY-based vectors. We generated a collection of silencing vectors based on 400 tomato (Solanum lycopersicum) expressed sequence tags in this TRV-LIC background. We have used this vector to identify roles for SlMADS1 and its Nicotiana benthamiana homologs, NbMADS4-1 and -2 in flowering. We find that NbMADS4-1 and -2 act nonredundantly in floral development and silencing of either gene results in loss of organ identity. This TRV-LIC vector should be a valuable resource to the plant community.

A ligation-independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development.

Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50 kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a prerecognition complex containing the p50 effector and NRIP1.

Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector.

Plasmodesmata (PD) transport developmentally important nucleic acids and proteins between plant cells. Primary PD form during cell division and are simple, linear channels. Secondary PD form in existing cell walls after cell division and are simple, twinned, or branched. PD function undergoes a marked reduction at the mid-torpedo stage of Arabidopsis embryogenesis. Two mutants, increased size exclusion limit (ise)1 and ise2, fail to undergo this transition, and their null mutations are embryonically lethal. We investigated the ultrastructure of PD in early-, mid-, and late-torpedo-stage embryos and in young leaves. Wild-type (WT) embryos contain twinned and branched (T/B) PD at all stages, but ise1 and ise2 embryos contain significantly higher proportions of T/B PD than WT embryos. WT T/B PD formation occurs in a stage- and tissue-specific pattern that is reversed in ise1 embryos. Silencing ISE1 in Nicotiana benthamiana leaves increases the frequency of secondary PD in existing cell walls. Silencing ISE2 increases the proportion of T/B secondary PD formed. Silenced tissues exhibit increased PD-mediated movement of green fluorescent protein tracers. Thus, silencing of ISE1 and ISE2 phenocopies ise1 and ise2 mutant embryos: when wild-type ISE1 and ISE2 functions are lost, de novo production of PD occurs, leading to increased intercellular transport.

Loss of INCREASED SIZE EXCLUSION LIMIT (ISE)1 or ISE2 increases the formation of secondary plasmodesmata.

&#x80DE;&#x95F4;&#x8FDE;&#x4E1D; (PD) &#x7ED3;&#x6784;&#x548C;&#x529F;&#x80FD;&#x4E0D;&#x540C;&#x690D;&#x7269;&#x53D1;&#x5C55;&#x7684;&#x6240;&#x6709;&#x9636;&#x6BB5;&#x7684;&#x65F6;&#x95F4;&#x4E0A;&#x548C;&#x7A7A;&#x95F4;&#x4E0A;&#x3002;&#x5C40;&#x90E8;&#x653E;&#x7535;&#x671F;&#x95F4;&#xFF0C;&#x6E90;&#x81EA;&#x6216;&#x90AE;&#x5BC4;&#xFF0C;&#x7EC6;&#x80DE;&#x5206;&#x88C2;&#x88AB;&#x6307;&#x5B9A;&#x4E3A;&#x5C0F;&#x5B66;&#x6216;&#x4E2D;&#x5B66;&#x6839;&#x636E;&#x53E4;&#x5178;&#x672F;&#x8BED;&#x3002;PD &#x7ED3;&#x6784;&#x53EF;&#x80FD;&#x7B80;&#x5355;&#xFF0C;&#x59D0;&#x59B9;&#xFF0C;&#x6216;&#x5206;&#x652F;&#x3002;PD &#x53F6;&#x3001; &#x6839;&#x3001; &#x548C;&#x80DA;&#x80CE;&#x53D1;&#x80B2;&#x8FC7;&#x7A0B;&#x7684;&#x7814;&#x7A76;&#x5DF2;&#x5BFC;&#x81F4;&#x6B20;&#x6210;&#x719F;&#x7684;&#x7EC4;&#x7EC7;&#x4E2D;&#x7684;&#x5355;&#x5143;&#x683C;&#x5305;&#x542B;&#x4E3B;&#x8981;&#x662F;&#x7B80;&#x5355;&#x7684; PD &#x7684;&#x6CDB;&#x5316;&#x3002;&#x65B0;&#x7684;&#x91CF;&#x5316;&#x5206;&#x6790;&#x63ED;&#x793A;&#x59D0;&#x59B9;&#x548C;&#x652F;&#x94FE; PD &#x8FD8;&#x672A;&#x6210;&#x719F;&#x7684;&#x7EC4;&#x7EC7;&#x4E2D;&#x4F1A;&#x51FA;&#x73B0;&#x3002;&#x65B0;&#x7684;&#x6570;&#x636E;&#x8FD8;&#x7A81;&#x51FA;&#x4F5C;&#x4E3A;&#x6807;&#x8BB0;&#x4E3A;&#x672A;&#x6210;&#x719F;&#x7EC4;&#x7EC7;&#x4EE5;&#x53CA; PD &#x8D34;&#x6807; PD&#xFF0C;&#x6210;&#x719F;&#x7684;&#x7EC4;&#x7EC7;&#x4E2D;&#x75C5;&#x6BD2;&#x8FD0;&#x52A8;&#x86CB;&#x767D;&#x7684;&#x591A;&#x529F;&#x80FD;&#x6027;&#x3002;&#x6458;&#x8981;&#x7684;&#x5F62;&#x6210;&#x548C;&#x529F;&#x80FD;&#x7684;&#x5C0F;&#x5B66;&#x3001; &#x4E2D;&#x5B66;&#x548C;&#x5206;&#x652F; PD &#x53F6;&#x3001; &#x6BDB;&#x3001; &#x80DA;&#x80CE;&#x3001; &#x830E;&#x5C16;&#x8131;&#x6BD2;&#x3001; &#x7EF4;&#x548C;&#x6839;&#x7CFB;&#x53D1;&#x80B2;&#x671F;&#x95F4;&#x5F3A;&#x8C03;&#x4ECB;&#x5BFC; PD &#x7684;&#x51FA;&#x8272;&#x548C;&#x4E0D;&#x53EF;&#x6216;&#x7F3A;&#x7684;&#x7279;&#x5B9A;&#x690D;&#x7269;&#x7684;&#x7EC6;&#x80DE;&#x95F4;&#x901A;&#x8BAF;&#x7CFB;&#x7EDF;&#x3002;

&#x80DE;&#x95F4;&#x8FDE;&#x4E1D;&#x5728;&#x5F00;&#x53D1;&#x671F;&#x95F4;&#xFF1A; &#x91CD;&#x65B0;&#x5BA1;&#x67E5;&#x7684;&#x5C0F;&#x5B66;&#x3001; &#x4E2D;&#x5B66;&#x548C;&#x5206;&#x652F;&#x7684;&#x80DE;&#x95F4;&#x8FDE;&#x4E1D;&#x7ED3;&#x6784;&#x4E0E;&#x529F;&#x80FD;&#x7684;&#x91CD;&#x8981;&#x6027;&#x3002;

Plasmodesmata (PD) structure and function vary temporally and spatially during all stages of plant development. PD that originate during, or post, cell division are designated as primary or secondary according to classical terminology. PD structure may be simple, twinned, or branched. Studies of PD during leaf, root, and embryo development have lead to the generalization that cells in less mature tissues contain predominantly simple PD. New quantitative analyses reveal that twinned and branched PD also occur in immature tissues. New data also highlight the versatility of viral movement proteins as tags for labeling PD in immature tissues as well as PD in mature tissues. A summary of the formation and function of primary, secondary, and branched PD during leaf, trichome, embryo, apical meristem, vascular cambium, and root development underscores the remarkable and indispensible plant-specific intercellular communication system that is mediated by PD.

Plasmodesmata during development: re-examination of the importance of primary, secondary, and branched plasmodesmata structure versus function.

Fonction et structure des plasmodesmes (PD) varient dans le temps et dans l&#39;espace durant toutes les &eacute;tapes du d&eacute;veloppement des plantes. PD qui sont cr&eacute;&eacute;s au cours, ou par la poste, la division cellulaire sont d&eacute;sign&eacute;s comme primaire ou secondaire, selon la terminologie classique. Structure de PD peut &ecirc;tre simple, jumel&eacute;, ou ramifi&eacute;s. &Eacute;tudes de PD au cours du d&eacute;veloppement embryonnaire feuille et racine ont conduit &agrave; la g&eacute;n&eacute;ralisation que les cellules dans les tissus moins matures contiennent principalement simple PD. Nouvelles analyses quantitatives r&eacute;v&egrave;lent que PD jumel&eacute;e et ramifi&eacute; s&#39;observent aussi dans les tissus immatures. Nouvelles donn&eacute;es soulignent aussi la polyvalence des prot&eacute;ines virales mouvement sous forme de balises pour l&#39;&eacute;tiquetage en tissus immatures ainsi que PD PD dans les tissus matures. Un r&eacute;sum&eacute; de la formation et de la fonction de PD primaire, secondaire et ramifi&eacute;e au cours de la feuille, trichome, embryon, m&eacute;rist&egrave;me apical, cambium vasculaire et le d&eacute;veloppement racinaire met en &eacute;vidence le syst&egrave;me de communication intercellulaire de remarquable et indispensable sp&eacute;cifiques &agrave; l&#39;installation qui est v&eacute;hicul&eacute; par le PD.

Plasmodesmes au cours du d&eacute;veloppement : r&eacute;examen de l&#39;importance de la structure primaire, secondaire et ramifi&eacute;s plasmodesmes fonction.

Plasmodesmata (PD) Struktur und Funktion unterscheiden sich zeitlich und r&auml;umlich w&auml;hrend aller Phasen der Pflanzenentwicklung. PD, die w&auml;hrend stammen oder buchen, Zellteilung werden als prim&auml;re oder sekund&auml;re nach klassischer Terminologie bestimmt. PD-Struktur kann einfach sein, eine St&auml;dtepartnerschaft oder verzweigt. Studien der PD aus Blatt, Wurzel und Embryo Entwicklung haben zu der Verallgemeinerung, dass Zellen in weniger ausgereiften Gewebe vorwiegend einfache PD enthalten. Neue Quantitative Analysen zeigen, dass Partnerst&auml;dten und verzweigte PD auch in unreifen Gewebe auftreten. Neue Daten zeigen auch die Vielseitigkeit der viralen Bewegung Proteine als Markierungen f&uuml;r die Bezeichnung PD in unreifen Gewebe sowie PD in Reife Gewebe. Eine &Uuml;bersicht &uuml;ber die Bildung und Funktion der prim&auml;ren, sekund&auml;ren und verzweigte PD w&auml;hrend Blatt Trichom, Embryo, Apical Meristem, vaskul&auml;re Kambium und Wurzel Entwicklung unterstreicht das bemerkenswerte und unverzichtbaren anlagenspezifischen interzellul&auml;re Kommunikation-System, das von PD vermittelt wird.

Plasmodesmata w&auml;hrend der Entwicklung: &Uuml;berpr&uuml;fung der Bedeutung der prim&auml;ren, sekund&auml;ren und verzweigte Plasmodesmata Struktur versus-Funktion.

Funzione e struttura Plasmodesmi (PD) variano temporalmente e spazialmente durante tutte le fasi di sviluppo di pianta. PD che provengono durante o post, divisione cellulare sono designati come primaria o secondaria secondo la terminologia classica. Struttura del PD pu&ograve; essere semplice, gemellata, o ramificata. Gli studi del PD durante lo sviluppo dell&#39;embrione, radice e foglia hanno portato la generalizzazione che le cellule nei tessuti meno maturi contengono prevalentemente semplice PD. Nuove analisi quantitative rivelano che PD gemellate e ramificata si verificano anche nei tessuti immaturi. Nuovi dati evidenziano anche la versatilit&agrave; delle proteine virali movimento come tag per l&#39;etichettatura di PD in tessuti immaturi come PD nei tessuti maturi. Una sintesi della formazione e funzione del PD primarie, secondarie e ramificati durante, foglia, tricomi, embrione, meristema apicale, del cambio vascolare e lo sviluppo delle radici sottolinea il sistema notevole e indispensabile pianta specifica comunicazione intercellulare che &egrave; mediato dal PD.

Plasmodesmi durante lo sviluppo: riesame dell&#39;importanza della struttura primaria, secondaria e ramificata Plasmodesmi versus funzione.

&#x539F;&#x5F62;&#x8CEA;&#x9023;&#x7D61; (PD) &#x306E;&#x69CB;&#x9020;&#x3068;&#x6A5F;&#x80FD;&#x7A7A;&#x9593;&#x30D7;&#x30E9;&#x30F3;&#x30C8;&#x958B;&#x767A;&#x306E;&#x3059;&#x3079;&#x3066;&#x306E;&#x6BB5;&#x968E;&#x306B;&#x3088;&#x3063;&#x3066;&#x7570;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x4E2D;&#x306B;&#x306F;&#x3001;&#x767A;&#x4FE1;&#x3057;&#x305F;&#x308A;&#x3001;&#x6295;&#x7A3F;&#x3001;&#x7D30;&#x80DE;&#x5206;&#x88C2;&#x306E; PD &#x306F;&#x3001;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x307E;&#x305F;&#x306F;&#x30BB;&#x30AB;&#x30F3;&#x30C0;&#x30EA;&#x53E4;&#x5178;&#x7684;&#x306A;&#x7528;&#x8A9E;&#x306B;&#x3088;&#x308B;&#x3068;&#x3068;&#x3057;&#x3066;&#x6307;&#x5B9A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x59C9;&#x59B9;&#x90FD;&#x5E02;&#x3001;&#x307E;&#x305F;&#x306F;&#x5206;&#x5C90; PD &#x69CB;&#x9020;&#x306F;&#x30B7;&#x30F3;&#x30D7;&#x30EB;&#x3067;&#x3042;&#x308B;&#x304C;&#x3002;PD &#x306E;&#x7814;&#x7A76;&#x8449;, &#x6839;, &#x304A;&#x3088;&#x3073;&#x80DA;&#x306E;&#x958B;&#x767A;&#x4E2D;&#x306F;&#x3042;&#x307E;&#x308A;&#x6210;&#x719F;&#x3057;&#x305F;&#x7D44;&#x7E54;&#x7D30;&#x80DE;&#x304C;&#x4E3B;&#x306B;&#x5358;&#x7D14;&#x306A; PD &#x3092;&#x542B;&#x3080;&#x6C4E;&#x5316;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x65B0;&#x3057;&#x3044;&#x5B9A;&#x91CF;&#x7684;&#x89E3;&#x6790;&#x53CC;&#x6676;&#x3068;&#x5206;&#x5C90;&#x3057;&#x305F; PD &#x3067;&#x672A;&#x719F;&#x306A;&#x7D44;&#x7E54;&#x3082;&#x767A;&#x751F;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x307E;&#x3059;&#x3002;&#x65B0;&#x3057;&#x3044;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x307E;&#x305F;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x79FB;&#x884C;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x591A;&#x69D8;&#x6027; PD &#x672A;&#x719F;&#x7D44;&#x7E54;&#x3068;&#x3057;&#x3066; PD &#x30E9;&#x30D9;&#x30EA;&#x30F3;&#x30B0;&#x6210;&#x719F;&#x3057;&#x305F;&#x7D44;&#x7E54;&#x306E;&#x305F;&#x3081;&#x306E;&#x30BF;&#x30B0;&#x3068;&#x3057;&#x3066;&#x5F37;&#x8ABF;&#x8868;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x3001;&#x30BB;&#x30AB;&#x30F3;&#x30C0;&#x30EA;&#x3001;&#x304A;&#x3088;&#x3073;&#x5206;&#x5C90;&#x306E; PD &#x8449;, &#x6BDB;&#x8338;&#x3001;&#x80DA;&#x3001;&#x9802;&#x7DAD;&#x7BA1;&#x675F;&#x5F62;&#x6210;&#x5C64;&#x53CA;&#x3073;&#x30EB;&#x30FC;&#x30C8;&#x958B;&#x767A;&#x4E2D;&#x306E;&#x6A5F;&#x80FD;&#x3068;&#x5F62;&#x6210;&#x306E;&#x6982;&#x8981; PD &#x306B;&#x3088;&#x3063;&#x3066;&#x5A92;&#x4ECB;&#x3055;&#x308C;&#x308B;&#x9855;&#x8457;&#x304A;&#x3088;&#x3073;&#x4E0D;&#x53EF;&#x6B20;&#x306A;&#x7279;&#x5B9A;&#x306E;&#x690D;&#x7269;&#x7D30;&#x80DE;&#x9593;&#x30B3;&#x30DF;&#x30E5;&#x30CB;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3; &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x5F37;&#x8ABF;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30D7;&#x30E9;&#x30B9;&#x30E2;&#x30C7;&#x30B9;&#x30E0;&#x958B;&#x767A;&#x4E2D;: &#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x3001;&#x30BB;&#x30AB;&#x30F3;&#x30C0;&#x30EA;&#x3001;&#x304A;&#x3088;&#x3073;&#x5206;&#x5C90;&#x30D7;&#x30E9;&#x30B9;&#x30E2;&#x30C7;&#x30B9;&#x30E0;&#x69CB;&#x9020;&#x5BFE;&#x95A2;&#x6570;&#x306E;&#x91CD;&#x8981;&#x6027;&#x306E;&#x518D;&#x691C;&#x8A0E;&#x3002;

Plasmodesmata (PD) &#xAD6C;&#xC870;&#xC640; &#xAE30;&#xB2A5; &#xC2DD;&#xBB3C; &#xBC1C;&#xB2EC;&#xC758; &#xBAA8;&#xB4E0; &#xB2E8;&#xACC4; &#xB3D9;&#xC548; temporally&#xACFC; &#xACF5;&#xAC04;&#xC744; &#xB2E4;&#xB985;&#xB2C8;&#xB2E4;. PD, &#xD558;&#xB294; &#xB3D9;&#xC548; &#xBC1C;&#xC0DD; &#xD55C; &#xB610;&#xB294; &#xAC8C;&#xC2DC;, &#xC138;&#xD3EC; &#xBD84;&#xC5F4; &#xB610;&#xB294; &#xD074;&#xB798;&#xC2DD; &#xC6A9;&#xC5B4;&#xC5D0; &#xB530;&#xB77C; &#xBCF4;&#xC870;&#xB85C; &#xC9C0;&#xC815; &#xB429;&#xB2C8;&#xB2E4;. &#xCD5C;&#xC120;&#xC744;, &#xB610;&#xB294; &#xBD84;&#xAE30; PD &#xAD6C;&#xC870; &#xAC04;&#xB2E8;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC78E;, &#xBFCC;&#xB9AC;, &#xADF8;&#xB9AC;&#xACE0; &#xBC30;&#xC544; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; PD&#xC758; &#xC5F0;&#xAD6C;&#xB294; &#xB35C; &#xC131;&#xC219;&#xD55C; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; &#xC138;&#xD3EC; &#xC8FC;&#xB85C; &#xAC04;&#xB2E8;&#xD55C; PD &#xD3EC;&#xD568; &#xD558;&#xB294; &#xC77C;&#xBC18;&#xD654;&#xB97C; &#xB9AC;&#xB4DC;. &#xC0C8;&#xB85C;&#xC6B4; &#xC815;&#xB7C9; &#xBD84;&#xC11D; &#xACF5;&#xAC1C; twinned &#xBD84;&#xAE30; PD&#xB3C4; &#xC5B4; &#xB824; &#xC11C; &#xC870;&#xC9C1;&#xC5D0; &#xBC1C;&#xC0DD;&#xD560;. &#xC0C8; &#xB370;&#xC774;&#xD130;&#xB294; &#xB610;&#xD55C; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC774;&#xB3D9; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xB2E4;&#xC591;&#xC131;&#xC73C;&#xB85C; &#xC131;&#xC219;&#xD55C; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; PD&#xB85C; &#xC11C; &#xBBF8; &#xC131;&#xC219;&#xD55C; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; PD&#xB97C; &#xB77C;&#xBCA8;&#xC5D0; &#xB300; &#xD55C; &#xD0DC;&#xADF8;&#xB97C; &#xAC15;&#xC870; &#xD45C;&#xC2DC; &#xD569;&#xB2C8;&#xB2E4;. &#xD615;&#xC131;&#xC758; &#xC694;&#xC57D; &#xBC0F; &#xC78E;, trichome, &#xBC30;&#xC544;, apical &#xBD84;&#xC5F4; &#xC870;&#xC9C1;, &#xD608;&#xAD00; cambium &#xBC0F; &#xB8E8;&#xD2B8; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; 1 &#xCC28;, 2 &#xCC28;, &#xADF8;&#xB9AC;&#xACE0; &#xBD84;&#xAE30; PD&#xC758; &#xAE30;&#xB2A5; &#xBC11;&#xC904; PD &#xC911;&#xC7AC; &#xD3C9;&#xBC94; &#xD558; &#xACE0; &#xD544;&#xC218;&#xC801;&#xC778; &#xC2DD;&#xBB3C; &#xD2B9;&#xC815; &#xC138;&#xD3EC; &#xD1B5;&#xC2E0; &#xC2DC;&#xC2A4;&#xD15C;.

&#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; plasmodesmata: &#xBE44;&#xAD50; &#xAE30;&#xB2A5; &#xAE30;&#xBCF8;, &#xBCF4;&#xC870; &#xBC0F; &#xBD84;&#xAE30; plasmodesmata &#xAD6C;&#xC870;&#xC758; &#xC911;&#xC694;&#xC131;&#xC744; &#xB2E4;&#xC2DC;.

Plasmodesmos (PD) estructura y funci&oacute;n var&iacute;an temporal y espacial durante todas las etapas de desarrollo de las plantas. PD que originan durante o post, divisi&oacute;n celular se se&ntilde;alan como primario o secundario seg&uacute;n la terminolog&iacute;a cl&aacute;sica. Estructura de PD puede ser simple, hermanadas, o ramificado. Estudios de la EP durante el desarrollo del embri&oacute;n, hojas y ra&iacute;z han dado lugar a la generalizaci&oacute;n de que las c&eacute;lulas en tejidos menos maduros contienen predominantemente simple PD. Nuevos an&aacute;lisis cuantitativos revelan que el PD maclado y ramificado tambi&eacute;n ocurrir en tejidos inmaduros. Nuevos datos tambi&eacute;n destacan la versatilidad de las prote&iacute;nas del movimiento viral como etiquetas para el etiquetado de PD en tejidos inmaduros como PD en tejidos maduros. Un Resumen de la formaci&oacute;n y funci&oacute;n de PD primario, secundario y ramificado en hoja, tricomas, embri&oacute;n, meristemo apical, cambium vascular y desarrollo radicular pone de relieve el sistema de comunicaci&oacute;n intercelular de la planta espec&iacute;fica notable e indispensable que es mediado por PD.

Plasmodesmos durante el desarrollo: revisi&oacute;n de la importancia de la estructura primaria, secundaria y ramificado plasmodesmos versus funci&oacute;n.

We use Arabidopsis thaliana embryogenesis as a model system for studying intercellular transport via plasmodesmata (PD). A forward genetic screen for altered PD transport identified increased size exclusion limit (ise) 1 and ise2 mutants with increased intercellular transport of fluorescent 10-kDa tracers. Both ise1 and ise2 exhibit increased formation of twinned and branched PD. ISE1 encodes a mitochondrial DEAD-box RNA helicase, whereas ISE2 encodes a DEVH-type RNA helicase. Here, we show that ISE2 foci are localized to the chloroplast stroma. Surprisingly, plastid development is defective in both ise1 and ise2 mutant embryos. In an effort to understand how RNA helicases that localize to different organelles have similar impacts on plastid and PD development/function, we performed whole-genome expression analyses. The most significantly affected class of transcripts in both mutants encode products that target to and enable plastid function. These results reinforce the importance of plastid-mitochondria-nucleus cross-talk, add PD as a critical player in the plant cell communication network, and thereby illuminate a previously undescribed signaling pathway dubbed organelle-nucleus-plasmodesmata signaling. Several genes with roles in cell wall synthesis and modification are also differentially expressed in both mutants, providing new targets for investigating PD development and function.

Organelle-nucleus cross-talk regulates plant intercellular communication via plasmodesmata.

Plant cells are surrounded by cellulosic cell walls, creating a potential challenge to resource sharing and information exchange between individual cells. To overcome this, plants have evolved channels called plasmodesmata that provide cytoplasmic continuity between each cell and its immediate neighbors. We first review plasmodesmata basics-their architecture, their origin, the types of cargo they transport, and their molecular components. The bulk of this review discusses the regulation of plasmodesmata formation and function. Historically, plasmodesmata research has focused intensely on uncovering regulatory or structural proteins that reside within or immediately adjacent to plasmodesmata. Recent findings, however, underscore that plasmodesmata are exquisitely sensitive to signals far removed from the plasmodesmal channel itself. Signals originating from molecules and pathways that regulate cellular homeostasis-such as reactive oxygen species, organelle-organelle signaling, and organelle-nucleus signaling-lead to astonishing alterations in gene expression that affect plasmodesmata formation and function.

Plasmodesmata paradigm shift: regulation from without versus within.

Maize and Arabidopsis thaliana class 1 reversibly glycosylated polypeptides (C1RGPs) are plasmodesmata-associated proteins. Previously, over-expression of Arabidopsis C1RGP AtRGP2 in Nicotiana tabacum was shown to reduce intercellular transport of photoassimilate, resulting in stunted, chlorotic plants, and inhibition of local cell-to-cell spread of tobacco mosaic virus (TMV). Here, we used virus induced gene silencing to examine the effects of reduced levels of C1RGPs in Nicotiana benthamiana. Silenced plants show wild-type growth and development. Intercellular transport in silenced plants was probed using fluorescently labeled TMV and its movement protein, P30. P30 shows increased cell-to-cell movement and TMV exhibited accelerated systemic spread compared to control plants. These results support the hypothesis that C1RGPs act to regulate intercellular transport via plasmodesmata.

Reduced levels of class 1 reversibly glycosylated polypeptide increase intercellular transport via plasmodesmata.

In plants, plasmodesmata (PD) serve as channels for micromolecular and macromolecular cell-to-cell transport. Based on structure, PD in immature tissues are classified into two types, simple and branched (X- and Y-shaped) or twinned. The maximum size of molecules capable of PD transport defines PD aperture, known as the PD size exclusion limit. Here we report an Arabidopsis mutation, decreased size exclusion limit1 (dse1), that exhibits reduced cell-to-cell transport of the small (524 Da) fluorescent tracer 8-hydroxypyrene-1,3,6-trisulfonic acid at the midtorpedo stage of embryogenesis. Correspondingly, the fraction of X- and Y-shaped and twinned PD was reduced in dse1 embryos compared with WT embryos at this stage, suggesting that the frequency of PD is related to transport capability. dse1 is caused by a point mutation in At4g29860 (previously termed TANMEI) at the last donor splice site of its transcript, resulting in alternative splicing in both the first intron and the last intron. AtDSE1 is a conserved eukaryotic 386-aa WD-repeat protein critical for Arabidopsis morphogenesis and reproduction. Similar to its homologs in mouse, null mutants are embryo-lethal. The weak loss-of-function mutant dse1 exhibits pleiotropic phenotypes, including retarded vegetative growth, delayed flowering time, dysfunctional male and female organs, and delayed senescence. Finally, silencing of DSE1 in Nicotiana benthamiana leaves leads to reduced movement of GFP fused to tobacco mosaic virus movement protein. Thus, DSE1 is important for regulating PD transport between plant cells.

Plasmodesmata formation and cell-to-cell transport are reduced in decreased size exclusion limit 1 during embryogenesis in Arabidopsis.

Delivery of dsRNA into Plants by VIGS Methodology.

Following the recognition of pathogen-encoded effectors, plant TIR-NB-LRR immune receptors induce defense signaling by a largely unknown mechanism. We identify a novel and conserved role for the SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-domain transcription factor SPL6 in enabling the activation of the defense transcriptome following its association with a nuclear-localized immune receptor. During an active immune response, the Nicotiana TIR-NB-LRR N immune receptor associates with NbSPL6 within distinct nuclear compartments. NbSPL6 is essential for the N-mediated resistance to Tobacco mosaic virus. Similarly, the presumed Arabidopsis ortholog AtSPL6 is required for the resistance mediated by the TIR-NB-LRR RPS4 against Pseudomonas syringae carrying the avrRps4 effector. Transcriptome analysis indicates that AtSPL6 positively regulates a subset of defense genes. A pathogen-activated nuclear-localized TIR-NB-LRR like N can therefore regulate defense genes through SPL6 in a mechanism analogous to the induction of MHC genes by mammalian immune receptors like CIITA and NLRC5.

Novel positive regulatory role for the SPL6 transcription factor in the N TIR-NB-LRR receptor-mediated plant innate immunity.

Plasmodesmata (PD) are channels that connect the cytoplasm of adjacent plant cells, permitting intercellular transport and communication. PD function and formation are essential to plant growth and development, but we still know very little about the genetic pathways regulating PD transport. Here, we present a method for assaying changes in the rate of PD transport following genetic manipulation. Gene expression in leaves is modified by virus-induced gene silencing. Seven to ten days after infection with Tobacco rattle virus carrying a silencing trigger, the gene(s) of interest is silenced in newly arising leaves. In these new leaves, individual cells are then transformed with Agrobacterium to express GFP, and the rate of GFP diffusion via PD is measured. By measuring GFP diffusion both within the epidermis and between the epidermis and mesophyll, the assay can be used to study the effects of silencing a gene(s) on PD transport in general, or transport through secondary PD specifically. Plant biologists working in several fields will find this assay useful, since PD transport impacts plant physiology, development, and defense.

Article	Year
Applications and advantages of virus-induced gene silencing for gene function studies in plants. The Plant journal : for cell and molecular biology\| PubMed ID: 15315635	2004
Mechanisms of plant resistance to viruses. Nature reviews. Microbiology\| PubMed ID: 16132037	2005
Efficient virus-induced gene silencing in Arabidopsis. Plant physiology\| PubMed ID: 16815951	2006
A novel role for the TIR domain in association with pathogen-derived elicitors. PLoS biology\| PubMed ID: 17298188	2007
The functions of plant TIR domains. Science's STKE : signal transduction knowledge environment\| PubMed ID: 17726177	2007
A ligation-independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development. Plant physiology\| PubMed ID: 17932306	2007
Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector. Cell\| PubMed ID: 18267075	2008
Loss of INCREASED SIZE EXCLUSION LIMIT (ISE)1 or ISE2 increases the formation of secondary plasmodesmata. Current biology : CB\| PubMed ID: 20434343	2010
Plasmodesmata during development: re-examination of the importance of primary, secondary, and branched plasmodesmata structure versus function. Protoplasma\| PubMed ID: 21174132	2011
Organelle-nucleus cross-talk regulates plant intercellular communication via plasmodesmata. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22106293	2011
Plasmodesmata paradigm shift: regulation from without versus within. Annual review of plant biology\| PubMed ID: 22136566	2012
Reduced levels of class 1 reversibly glycosylated polypeptide increase intercellular transport via plasmodesmata. Plant signaling & behavior\| PubMed ID: 22274744	2012
Plasmodesmata formation and cell-to-cell transport are reduced in decreased size exclusion limit 1 during embryogenesis in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22411811	2012
Delivery of dsRNA into Plants by VIGS Methodology. CSH protocols\| PubMed ID: 22485703	2006
Novel positive regulatory role for the SPL6 transcription factor in the N TIR-NB-LRR receptor-mediated plant innate immunity. PLoS pathogens\| PubMed ID: 23516366	2013
Investigating Plasmodesmata Genetics with Virus-Induced Gene Silencing and an Agrobacterium-Mediated GFP Movement Assay. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 25287205	2015

Tessa M. Burch-Smith

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