fluorescence-situ-hybridizations-fish-for-localization-viruses

Fluorescence <em>in situ</em> Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

Volcani Center

Grapevine virus A (GVA) is closely associated with the economically important rugose-wood disease of grapevine. In an attempt to develop GVA resistance, we made a GFP-tagged GVA-minireplicon and utilized it as a tool to consistently activate RNA silencing. Launching the GVA-minireplicon by agroinfiltration delivery resulted in a strong RNA silencing response. In light of this finding, we produced transgenic Nicotiana benthamiana plants expressing the GVA-minireplicon, which displayed phenotypes that could be attributed to reproducibly and consistently activate post-transcriptional gene silencing (PTGS). These included: (i) low accumulation of the minireplicon-derived transgene; (ii) low GFP expression that was increased upon agroinfiltration delivery of viral suppressors of silencing; and (iii) resistance against GVA infection, which was found in 60%, and in 90-95%, of T1 and T2 progenies, respectively. A grafting assay revealed that non-silenced scions exhibited GVA resistance when they were grafted onto silenced rootstocks, suggesting transmission of RNA silencing from silenced rootstocks to non-silenced scions. Despite being extremely resistant to GVA infection, the transgenic plants were susceptible to the closely related vitivirus, GVB. Furthermore, infection of the silenced plants with GVB or Potato virus Y (PVY) resulted in suppression of the GVA-specific defense. From these data we conclude that GVA-minireplicon-mediated RNA silencing provides an important and efficient approach for consistent activation of PTGS that can be used for controlling grapevine viruses. However, application of this strategy for virus resistance necessitates consideration of possible infection by other viruses.

Post-transcriptional gene silencing and virus resistance in Nicotiana benthamiana expressing a Grapevine virus A minireplicon.

A simple, rapid, inexpensive method for the localization of virus transcripts in plant and insect vector tissues is reported here. The method based on fluorescent in situ hybridization using short DNA oligonucleotides complementary to an RNA segment representing a virus transcript in the infected plant or insect vector. The DNA probe harbors a fluorescent molecule at its 5' or 3' ends. The protocol: simple fixation, hybridization, minimal washing and confocal microscopy, provides a highly specific signal. The reliability of the protocol was tested by localizing two phloem-limited plant virus transcripts in infected plants and insect tissues: Tomato yellow leaf curl virus (TYLCV) (Begomovirus: Geminiviridae), exclusively transmitted by the whitefly Bemisia tabaci (Gennadius) in a circulative non-propagative manner, and Potato leafroll virus (Polerovirus: Luteoviridae), similarly transmitted by the aphid Myzus persicae (Sulzer). Transcripts for both viruses were localized specifically to the phloem sieve elements of infected plants, while negative controls showed no signal. TYLCV transcripts were also localized to the digestive tract of B. tabaci, confirming TYLCV route of transmission. Compared to previous methods for localizing virus transcripts in plant and insect tissues that include complex steps for in-vitro probe preparation or antibody raising, tissue fixation, block preparation, sectioning and hybridization, the method described below provides very reliable, convincing, background-free results with much less time, effort and cost.

A simple, rapid and inexpensive method for localization of Tomato yellow leaf curl virus and Potato leafroll virus in plant and insect vectors.

Tomato yellow leaf curl virus (TYLCV) (Geminiviridae: Begomovirus) is exclusively vectored by the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). TYLCV transmission depends upon a 63-kDa GroEL protein produced by the vector's endosymbiotic bacteria. B. tabaci is a species complex comprising several genetically distinct biotypes that show different secondary-symbiont fauna. In Israel, the B biotype harbors Hamiltonella, and the Q biotype harbors Wolbachia and Arsenophonus. Both biotypes harbor Rickettsia and Portiera (the obligatory primary symbionts). The aim of this study was to determine which B. tabaci symbionts are involved in TYLCV transmission using B. tabaci populations collected in Israel. Virus transmission assays by B. tabaci showed that the B biotype efficiently transmits the virus, while the Q biotype scarcely transmits it. Yeast two-hybrid and protein pulldown assays showed that while the GroEL protein produced by Hamiltonella interacts with TYLCV coat protein, GroEL produced by Rickettsia and Portiera does not. To assess the role of Wolbachia and Arsenophonus GroEL proteins (GroELs), we used an immune capture PCR (IC-PCR) assay, employing in vivo- and in vitro-synthesized GroEL proteins from all symbionts and whitefly artificial feeding through membranes. Interaction between GroEL and TYLCV was found to occur in the B biotype, but not in the Q biotype. This assay further showed that release of virions protected by GroEL occurs adjacent to the primary salivary glands. Taken together, the GroEL protein produced by Hamiltonella (present in the B biotype, but absent in the Q biotype) facilitates TYLCV transmission. The other symbionts from both biotypes do not seem to be involved in transmission of this virus.

The transmission efficiency of tomato yellow leaf curl virus by the whitefly Bemisia tabaci is correlated with the presence of a specific symbiotic bacterium species.

The whitefly Bemisia tabaci is a cosmopolitan insect pest that harbors Portiera aleyrodidarum, the primary obligatory symbiotic bacterium, and several facultative secondary symbionts. Secondary symbionts in B. tabaci are generally associated with the bacteriome, ensuring their vertical transmission; however, Rickettsia is an exception and occupies most of the body cavity, except the bacteriome. The mode of Rickettsia transfer between generations and its subcellular localization in insect organs have not been investigated. Using electron and fluorescence microscopy, we show that Rickettsia infects the digestive, salivary, and reproductive organs of the insect; however, it was not observed in the bacteriome. Rickettsia invades the oocytes during early developmental stages and resides in follicular cells and cytoplasm; it is mostly excluded when the egg matures; however, some bacterial cells remain in the egg, ensuring their transfer to subsequent generations. Rickettsia was localized to testicles and the spermatheca, suggesting a horizontal transfer between males and females during mating. The bacterium was further observed at large amounts in midgut cells, concentrating in vacuole-like structures, and was located in the hemolymph, specifically at exceptionally large amounts around bacteriocytes and in fat bodies. Organs further infected by Rickettsia included the primary salivary glands and stylets, sites of possible secretion of the bacterium outside the whitefly body. The close association between Rickettsia and the B. tabaci digestive system might be important for digestive purposes. The vertical transmission of Rickettsia to subsequent generations occurs via the oocyte and not, like other secondary symbionts, the bacteriome.

Transovarial transmission of Rickettsia spp. and organ-specific infection of the whitefly Bemisia tabaci.

&quot;&#x79CD;&#x7C7B; Portiera aleyrodidarum&quot;&#x662F;&#x9884;&#x7559;&#x7684;&#x4E3B; endosymbiotic &#x83CC;&#x682A;&#x7684;&#x8671;&#xFF0C;&#x5305;&#x62EC;&#x7518;&#x85AF;&#x7C89;&#x8671;&#x70DF;&#x7C89;&#x8671;&#xFF0C;&#x5E76;&#x5411;&#x5176;&#x4E3B;&#x673A;&#x63D0;&#x4F9B;&#x5FC5;&#x9700;&#x8425;&#x517B;&#x7D20;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x8FD9;&#x79CD;&#x7EC6;&#x83CC;&#x4ECE; B &#x548C; Q B.&#x7C89;&#x8671;&#x751F;&#x7269;&#x578B;&#x7684;&#x4E24;&#x4E2A;&#x5B8C;&#x6574;&#x7684;&#x57FA;&#x56E0;&#x7EC4;&#x7684;&#x5E8F;&#x5217;&#x3002;

&#x4E3B; Endosymbiont&quot;&#x79CD;&#x7C7B; Portiera aleyrodidarum&quot;&#x5728; B &#x70DF;&#x7C89;&#x8671;&#x548C; Q &#x751F;&#x7269;&#x7684;&#x57FA;&#x56E0;&#x7EC4;&#x5E8F;&#x5217;&#x3002;

"Candidatus Portiera aleyrodidarum" is the obligate primary endosymbiotic bacterium of whiteflies, including the sweet potato whitefly Bemisia tabaci, and provides essential nutrients to its host. Here we report two complete genome sequences of this bacterium from the B and Q biotypes of B. tabaci.

Genome Sequences of the Primary Endosymbiont "Candidatus Portiera aleyrodidarum" in the Whitefly Bemisia tabaci B and Q Biotypes.

&laquo; Candidatus Portiera aleyrodidarum &raquo; est la bact&eacute;rie endosymbiotique primaires obligatoires des aleurodes, y compris la patate mouche blanche Bemisia tabaci et fournit des nutriments essentiels &agrave; son h&ocirc;te. Nous pr&eacute;sentons deux s&eacute;quences compl&egrave;te du g&eacute;nome de cette bact&eacute;rie de biotypes B et Q de b. tabaci.

S&eacute;quences du g&eacute;nome de l&#39;endosymbiote primaire &laquo; Candidatus Portiera aleyrodidarum &raquo; dans la mouche blanche Bemisia tabaci B et Q Biotypes.

&quot;Candidatus Portiera Aleyrodidarum&quot; ist die obligat prim&auml;re Endosymbiotic Bakterium der Whiteflies, einschlie&szlig;lich die S&uuml;&szlig;kartoffel Mottenschildl&auml;use Bemisia Tabaci, und bietet wesentliche N&auml;hrstoffe auf seinem Wirt. Hier berichten wir zwei komplette Genomsequenzen dieses Bakterium aus der B und Q Biotypen von B. Tabaci.

Genomsequenzen der prim&auml;re Endosymbionten &quot;Candidatus Portiera Aleyrodidarum&quot; in der Mottenschildl&auml;use Bemisia Tabaci B und Q Biotypen.

&quot;Candidatus Portiera aleyrodidarum&quot; &egrave; il batterio obligate endosimbiosi primaria di aleurodidi, tra cui la patata dolce aleurodidi Bemisia tabaci e fornisce le sostanze nutrienti essenziali al suo ospite. Qui riportiamo due sequenze completa del genoma di questo batterio dalla B e Q biotipi di b. tabaci.

Sequenze di genoma di endosimbiosi primaria &quot;Candidatus Portiera aleyrodidarum&quot; nell&#39;aleurodidi Bemisia tabaci B e Q biotipi.

&#x300C;&#x30AB;&#x30F3;&#x30AD;&#x30C4;&#x30B0;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x75C5; Portiera aleyrodidarum&quot;&#xFF05;30 &#x306E;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x5171;&#x751F;&#x30D0;&#x30AF;&#x30C6;&#x30EA;&#x30A2; &#x30B5;&#x30C4;&#x30DE;&#x30A4;&#x30E2; &#x30B3;&#x30CA;&#x30B8;&#x30E9;&#x30DF; &#x30BF;&#x30D0;&#x30B3;&#x30B3;&#x30CA;&#x30B8;&#x30E9;&#x30DF;, &#x3092;&#x542B;&#x3080;&#x30B3;&#x30CA;&#x30B8;&#x30E9;&#x30DF;&#x306E;&#x3067;&#x3042;&#x308A;&#x3001;&#x305D;&#x306E;&#x30DB;&#x30B9;&#x30C8;&#x306B;&#x5FC5;&#x8981;&#x306A;&#x6804;&#x990A;&#x7D20;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F; 2 &#x3064;&#x306E;&#x5B8C;&#x5168;&#x306A;&#x30B2;&#x30CE;&#x30E0; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3053;&#x306E;&#x83CC; B &#x304A;&#x3088;&#x3073; Q &#x30D0;&#x30A4;&#x30AA;&#x30BF;&#x30A4;&#x30D7; B. &#x30BF;&#x30D0;&#x30B3;&#x30B3;&#x30CA;&#x30B8;&#x30E9;&#x30DF;&#x304B;&#x3089;&#x306E;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x5171;&#x751F;&#x30BF;&#x30D0;&#x30B3;&#x30B3;&#x30CA;&#x30B8;&#x30E9;&#x30DF; B &#x306E;&#x300C;&#x30AB;&#x30F3;&#x30AD;&#x30C4;&#x30B0;&#x30EA;&#x30FC;&#x30CB;&#x30F3;&#x30B0;&#x75C5; Portiera aleyrodidarum&quot;&#x3068;&#x30D0;&#x30A4;&#x30AA;&#x30BF;&#x30A4;&#x30D7; Q &#x306E;&#x30B2;&#x30CE;&#x30E0;&#x5869;&#x57FA;&#x914D;&#x5217;&#x3002;

&quot;Candidatus Portiera aleyrodidarum&quot; whiteflies, &#xACE0;&#xAD6C;&#xB9C8; whitefly Bemisia tabaci&#xB97C; &#xD3EC;&#xD568; &#xD558; &#xC5EC; &#xC758;&#xBB34; &#xAE30;&#xBCF8; endosymbiotic &#xBC15;&#xD14C;&#xB9AC;&#xC544; &#xC774;&#xBA70; &#xADF8; &#xD638;&#xC2A4;&#xD2B8;&#xC5D0; &#xD544;&#xC218; &#xC601;&#xC591;&#xC18C;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xB294; B. tabaci&#xC758; B&#xC640; Q biotypes&#xC5D0;&#xC11C;&#xC774; &#xBC15;&#xD14C;&#xB9AC;&#xC544;&#xC758; &#xB450; &#xC644;&#xC804; &#xD55C; &#xAC8C;&#xB188; &#xC2DC;&#xD000;&#xC2A4;&#xB97C; &#xBCF4;&#xACE0;&#xD569;&#xB2C8;&#xB2E4;.

&#xAE30;&#xBCF8; Endosymbiont &quot;Candidatus Portiera aleyrodidarum&quot; Whitefly Bemisia tabaci B Q Biotypes&#xC758; &#xAC8C;&#xB188; &#xC2DC;&#xD000;&#xC2A4;.

&quot;Candidatus Portiera aleyrodidarum&quot; es la bacteria endosimbi&oacute;tica primaria obligan de mosquitas blancas, incluyendo la mosca blanca Bemisia tabaci de patata dulce y provee nutrientes esenciales a su anfitri&oacute;n. Presentamos dos secuencias del genoma completo de esta bacteria de los biotipos B y Q de B. tabaci.

Secuencias del genoma de la primaria Endosymbiont &quot;Candidatus Portiera aleyrodidarum&quot; en la mosca blanca Bemisia tabaci B y Q biotipos.

"Candidatus Portiera aleyrodidarum" is the primary endosymbiont of whiteflies. We report two complete genome sequences of this bacterium from the worldwide invasive B and Q biotypes of the whitefly Bemisia tabaci. Differences in the two genome sequences may add insights into the complex differences in the biology of both biotypes.

Article	Year
Post-transcriptional gene silencing and virus resistance in Nicotiana benthamiana expressing a Grapevine virus A minireplicon. Transgenic research\| PubMed ID: 18821075	2009
A simple, rapid and inexpensive method for localization of Tomato yellow leaf curl virus and Potato leafroll virus in plant and insect vectors. Journal of virological methods\| PubMed ID: 19406154	2009
The transmission efficiency of tomato yellow leaf curl virus by the whitefly Bemisia tabaci is correlated with the presence of a specific symbiotic bacterium species. Journal of virology\| PubMed ID: 20631135	2010
Transovarial transmission of Rickettsia spp. and organ-specific infection of the whitefly Bemisia tabaci. Applied and environmental microbiology\| PubMed ID: 22660706	2012
Genome Sequences of the Primary Endosymbiont "Candidatus Portiera aleyrodidarum" in the Whitefly Bemisia tabaci B and Q Biotypes. Journal of bacteriology\| PubMed ID: 23144417	2012
Comparison of the genome sequences of "Candidatus Portiera aleyrodidarum" primary endosymbionts of the whitefly Bemisia tabaci B and Q biotypes. Applied and environmental microbiology\| PubMed ID: 23315735	2013

Marina Brumin

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