Downregulation of dystrophin expression in pupae of the whitefly Bemisia tabaci inhibits the emergence of adults.

The whitefly Bemisia tabaci is a major pest to agriculture. Adults are able to fly for long distances and to colonize staple crops, herbs and ornamentals, and to vector viruses belonging to several important taxonomic groups. During their early development, whiteflies mature from eggs through several nymphal stages (instars I to IV) until adults emerge from pupae. We aim at reducing whitefly populations by inhibiting the emergence of adults from nymphs. Here we targeted dystrophin, a conserved protein essential for the development of the muscle system in humans, other animals and insects. We have exploited the fact that whitefly nymphs developing on tomato leaves feed from the plant phloem via their stylets. Thus, we delivered dystrophin-silencing double-stranded RNA to nymphs developing on leaves of tomato plantlets with their roots bathing in the silencing solution. Downregulation of dystrophin expression occurred mainly in pupae. Dystrophin silencing induced also the downregulation of the dystrophin-associated protein genes actin and tropomyosin, and disrupted F-actin. Most significantly, the treatment inhibited the emergence of adults from pupae, suggesting that targeting dystrophin may help to restrain whitefly populations. This study demonstrates for the first time the important role of dystrophin in the development of a major insect pest to agriculture.

First Report of Recombinant Potato virus Y Strains Infecting Potato in Jordan.

Potato (Solanum tuberosum L.) is an important vegetable crop in Jordan, occupying second position after olives. In 2012, potatoes were planted on about 6,000 ha with a production of about 141,000 t (2). Potato virus Y (PVY) is a serious problem for potato production worldwide. Recombinant strains of the virus were reported to cause tuber necrotic ringspot disease (PTNRD) in many potato-growing regions of the world. In the last few years, a new recombinant PVYNTN-NW that belongs to PVYZ (3) has been reported in the neighboring Syria. It included three recombination patterns, SYR-I, SYR-II, and SYR-III, and caused severe PTNRD (1). Since PVY is easily transmitted from one region to another by aphid vectors and infected potato seeds, this study was initiated to investigate the possible occurrence of PVY strains in Jordan. In October 2013, 33 leaf samples were collected from symptomatic potato plants cv. Spunta from Wadi Rum, Jordan (GPS coordinates 29°31'37.76″ N, 35°42'48.75″ E), the largest potato-producing area in Jordan. Sampled plants displayed leaf mottling and yellowing, symptoms similar to those caused by PVY. All samples were tested for PVY by DAS-ELISA using the ELISA kit (monoclonal cocktail) developed by BIOREBA (Reinach, Switzerland) to detect all PVY isolates. Twenty-nine samples were found positive for PVY by ELISA. To confirm virus infection, total RNA was extracted from all ELISA-positive samples and used as template in uniplex RT-PCR using strain-specific primers (1). The band pattern of PCR amplicons showed that 12 samples were infected with PVYNTN-NW genotype SYR-III and produced bands of 1,085, 441, and 278 bp. One sample was infected with PVYNTN (A) and produced bands of 1,307, 633, and 441 bp, and one other sample was infected with PVYNTN-NW genotype SYR-II and produced bands of 1,085 and 441 bp. Mixed infection with PVYNTN-NW genotype SYR-III and PVYNTN (B) was also detected in one sample producing bands of 278, 441, 1,085, and 1,307 bp. To confirm infection with the recombinant strains, PCR fragments of 278 bp amplified from three samples and 1,085 bp obtained from another three samples were directly sequenced and sequences were deposited in GenBank under accession numbers KJ159968, KJ159969, and KJ159970 for the 278-bp fragment and KJ159974, KJ159975, and KJ159976 for the 1,085-bp fragment. Sequence comparison with other PVY strains available in the NCBI database showed that the 278-bp fragment had the highest nucleotide sequence identity (100%) with PVY isolates SYR-III-A26 (AB461467) and SYR-III-2-4 (AB461457) from Syria. BLAST searches also showed that the 1,085-bp fragment shared 99% nucleotide identities with PVY isolates SYR-II-L3 (AB461482) and SYR-II-Be4 (AB461474) from Aleppo, Syria. To our knowledge, this is the first report of PVY recombinants in Jordan, and the first report of PVYNTN-NW recombinants infecting potato crop outside Syria. Since Europe is the main supplier of potato seeds for farmers in Jordan and Syria, the introduction of PVYNTN-NW to the region could have happened through infected potato seeds. Results of this study create new challenges for potato growers in Jordan as well as other countries in the region. References: (1) M. Chikh Ali et al. J. Virol. Methods 165:15, 2010. (2) FAO. http://faostat.fao.org/ (3) A. V. Karasev and S. M. Gray. Ann. Rev. Phytopathol. 51:571, 2013.

Studies on the biosynthesis of neurofilament proteins.

To determine whether the triplet polypeptides of neurofilaments arise by degradation of precursor, we studied the biosynthesis of neurofilament polypeptides both in vivo and in cell-free systems. Neurofilament-enriched fractions and polyribosomes were prepared from the same rabbit spinal cord homogenates. At 1 h after intracisternal administration of [34S]methionine, radiolabeled neurofilament proteins were detected in spinal cord homogenates as well as in isolated filaments. When polyribosomes from rabbit spinal cord were allowed to incorporate [35S]methionine into protein, triplet polypeptides were among the proteins labeled. Addition of spinal cord polyribosomes to rabbit reticulocyte lysates led to several cycles of translation of the spinal cord mRNA; the three neurofilament polypeptides were among the proteins synthesized in this system. The results demonstrate that the triplet polypeptides of neurofilaments are synthesized as such in the course of individual translational events and do not arise from degradation of P200 or a larger precursor.

The gene and the pseudogene for mouse p53 cellular tumor antigen are located on different chromosomes.

The chromosomal assignments of the two genes encoding the murine p53 cellular tumor antigen were determined by using a panel of mouse-Chinese hamster somatic cell hybrid clones and a mouse p53-specific cDNA clone. One gene, probably the functional member of the family, was found to be on chromosome 11. The other gene, which is probably a processed pseudogene, was assigned to chromosome 14. The potential relevance of these findings to documented cases of chromosome 11 trisomy are also discussed.

Acquisition and transmission of Agrobacterium by the whitefly Bemisia tabaci.

Whiteflies transmit many different plant pathogens. We show here that the whitefly Bemisia tabaci can also acquire Agrobacterium tumefaciens from liquid cultures and from crown galls, and transmit it to plants. Cloned tomato yellow leaf curl virus (TYLCV) DNA and A. tumefaciens tumor-inducing functions were used as reporter genes. Whiteflies were fed through membranes on cultures of Agrobacterium At::pTY4 containing a dimeric copy of an infectious TYLCV genomic clone between the T-DNA borders of a binary vector. One hour of feeding was sufficient for the insects to be able to transmit TYLCV to tomato test plants. Infectious Agrobacterium was recovered from whiteflies that fed on At::pTY4 cultures, indicating that bacteria was acquired and remained intact in the insects. Whiteflies also acquired the virulent A. tumefaciens strain C58 from crown galls and induced tumors in tomato test plants. PCR and Southern blot analyses indicated that the target plant tissues were transformed. These results show that an insect can transfer foreign genes to plants by acquiring and delivering transforming bacteria.

Isolation and characterization of free and membrane-bound polyribosomes from rabbit spinal cord.

A procedure is described for the preparation of free and membrane-bound polyribosomes from rabbit spinal cord. First, myelin and filaments are floated away from other subcellular components. The latter are then fractionated by differential centrifugation followed by sedimentation through a discontinuous sucrose gradient. The ribosomal fractions are characterized by their electron microscopic appearance, RNA/protein ratios and sedimentation profile in a linear sucrose gradient. Both membrane-bound and free polyribosomes are active in incorporating amino acids in a cell-free system, whether heterologous or homologous pH 5 enzyme fractions are employed. Autoradiographic analysis of translation products separated by electrophoresis on SDS-polyacrylamide gels shows that both ribosome fractions are active in the synthesis of a variety of proteins including polypeptides which comigrate with alpha- and beta-tubulins and actin. The utility of these polyribosome preparations for the cell-free study of protein biosynthesis is indicated by the high molecular weight of many of the translation products, and by the similarity of the electrophoretic pattern of translation products from the cell-free systems to the pattern of radioactive protein synthesized by rabbit spinal cord during the hour prior to sacrifice.

Similarity of neurofilament proteins from different parts of the rabbit nervous system.

In the nervous system, the various populations of neurons perform a large spectrum of functions. Although neurofilaments are a major constituent of the different neurons, the neurofilament protein composition and the expression of the genes specifying these proteins may not be the same throughout the entire nervous system. To investigate these two aspects of the biology of neurofilaments, we have prepared neurofilament-rich fractions from different regions of the nervous system of strains of rabbits known to present a genetically determined polymorphism involving one of the neurofilament polypeptides (P200). Filaments were isolated from brain, spinal cord, sciatic, optic and trigeminal nerves, and lumbar ventral and dorsal roots by a procedure not involving axonal flotation and yielding material suitable for comparative analysis within a single animal. The filaments were compared for their variability as a function of the region from which they were prepared. For any given animal, the neurofilament peptides migrate to identical positions on SDS-gel electropherograms. Whatever allele of P200 is expressed in filaments from one region, the same allele is also expressed in all of the other filament preparations from that animal. On two-dimensional analysis isomorphs of the P68 neurofilament protein are not present in the same amounts in different regions of the nervous system. These results indicate that, although it seems that the gene for the P200 neurofilament protein is expressed uniformly throughout the nervous system, there may be some topographic specificity in the distribution of the other constituent proteins of neurofilaments.

Digestive, salivary, and reproductive organs of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) B type.

A microscopic analysis of the morphology and ultrastructure of the digestive, salivary, and reproductive systems of adult Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) B type was conducted using light, scanning, and transmission electron microscopy. The internal anatomy of B. tabaci was found to be similar to that reported for Trialeurodes vaporariorum. In a microscopic analysis of the salivary glands, we have shown that each primary salivary gland is composed of at least 13 cells varying in morphology and staining differentially, while the accessory salivary glands are composed of four morphologically similar cells. We analyzed the course of the alimentary canal in B. tabaci, demonstrated the internal morphology of the organs, and clarified the location of the filter chamber relative to other organs in the whitefly. Our observations confirm that the pair of structures extending from the connecting chamber are caeca that may aid in fluid movement through the midgut and are not Malpighian tubules, as previously suggested. We confirm an earlier finding that the whitefly lacks Malpighian tubules, having instead specialized Malpighian-like cells within the filter chamber at the juncture with the internal ileum. Finally, we provide the first scanning electron microscopic analysis showing the reproductive organs of B. tabaci. Our investigation provides clarified terminology for several components of the digestive and excretory system. We also provide drawings and micrographs that will aid future researchers in localizing the internal organs of B. tabaci. We expect our analysis to provide a valuable tool for studying B. tabaci / plant virus interactions and physiological and biological aspects of this insect.

Tomato Breeding Lines Resistant and Tolerant to Tomato Yellow Leaf Curl Virus Issued from Lycopersicon hirsutum.

ABSTRACT Two tomato yellow leaf curl virus (TYLCV)-resistant plants from accessions LA1777 and LA386 of the wild tomato species Lycopersicon hirsutum have been crossed. The resulting resistant F1 plants were crossed with the domesticated tomato L. esculentum, and a series of selfing was performed. At each generation, individuals were selected for resistance (no symptoms and undetectable viral DNA) and tolerance (no symptoms but with detectable viral DNA) following controlled massive and repeated inoculations with viruliferous whiteflies. A stable BC1F4 line (denominated 902) that does not segregate for resistance was obtained. This line does not support virus accumulation, even upon extensive whitefly-mediated inoculation of young seedlings, and does not need protection with nets or insecticides. Another stable BC1F4 line (denominated 908) was tolerant to the virus. Both lines have good horticultural characteristics and bear 80- to 120-g red fruits. Analysis of segregation of susceptibility, tolerance, and resistance during the BC1F1 to BC1F4 crosses indicated that tolerance is controlled by a dominant major gene and resistance by two to three additive recessive genes. The resistant and tolerant lines do not need to be protected by insecticides or nets.

Rate of Tomato yellow leaf curl virus Translocation in the Circulative Transmission Pathway of its Vector, the Whitefly Bemisia tabaci.

ABSTRACT Whiteflies (Bemisia tabaci, biotype B) were able to transmit Tomato yellow leaf curl virus (TYLCV) 8 h after they were caged with infected tomato plants. The spread of TYLCV during this latent period was followed in organs thought to be involved in the translocation of the virus in B. tabaci. After increasing acquisition access periods (AAPs) on infected tomato plants, the stylets, the head, the midgut, a hemolymph sample, and the salivary glands dissected from individual insects were subjected to polymerase chain reaction (PCR) without any treatment; the presence of TYLCV was assessed with virus-specific primers. TYLCV DNA was first detected in the head of B. tabaci after a 10-min AAP. The virus was present in the midgut after 40 min and was first detected in the hemolymph after 90 min. TYLCV was found in the salivary glands 5.5 h after it was first detected in the hemolymph. Subjecting the insect organs to immunocapture-PCR showed that the virus capsid protein was in the insect organs at the same time as the virus genome, suggesting that at least some TYLCV translocates as virions. Although females are more efficient as vectors than males, TYLCV was detected in the salivary glands of males and of females after approximately the same AAP.

Analysis of myogenesis with recombinant DNA techniques.

Recombinant phages containing the rat skeletal muscle alpha-actin gene and the cytoplasmic beta-actin gene were isolated and the structure of these genes was determined. Both genes contain a large intron in the 5' untranslated region and smaller introns at codons 41, 267 and 327. In addition, the alpha-actin contains introns at codons 150 and 204 not present in the beta-actin gene, whereas the beta-actin gene contains an intron at codon 121. The evolutionary aspects of these findings are discussed. Active genes are organized in chromatin in a conformation which renders them preferentially sensitive to digestion with nucleolytic enzymes. The DNAase I sensitivity of genes programmed to be expressed during myogenesis was tested in a cloned cell population of a myogenic cell line. It was found that these genes are not preferentially sensitive to DNAase I in the chromatin of proliferating mononucleated cells. They become DNAase I sensitive during terminal differentiation.

Transgenic tomato plants expressing the tomato yellow leaf curl virus capsid protein are resistant to the virus.

The tomato yellow leaf curl virus (TYLCV) gene that encodes the capsid protein (V1) was placed under transcriptional control of the cauliflower mosaic virus 35S promoter and cloned into an Agrobacterium Ti-derived plasmid and used to transform plants from an interspecific tomato hybrid, Lycopersicon esculentum X L. pennellii (F1), sensitive to the TYLCV disease. When transgenic F1 plants, expressing the V1 gene, were inoculated with TYLCV using whiteflies fed on TYLCV-infected plants, they responded either as untransformed tomato or showed expression of delayed disease symptoms and recovery from the disease with increasingly more resistance upon repeated inoculation. Transformed plants that were as sensitive to inoculation as untransformed controls expressed the V1 gene at the RNA level only. All the transformed plants that recovered from disease expressed the TYLCV capsid protein.

Sequencing and assembly of a full-length infectious clone of grapevine virus B and its infectivity on herbaceous plants.

Grapevine virus B (GVB) has been found associated with corky bark-diseased vines. Although the sequence of a 7.6-kb cDNA clone from a GVB isolate from Italy has been described, striking differences in sequences between GVB isolates prompted us to construct an additional full-length GVB clone from the isolate 94/971 and to determine its complete sequence. The cDNA of GVB 94/971 shared a nucleotide sequence identity of only 77% with the GVB isolate from Italy. The cDNA of GVB 94/971 was infectious on Nicotiana plants as demonstrated by symptoms and by means of Northern blot, Western blot and electron microscopic analyses.

Making a friend from a foe: expressing a GroEL gene from the whitefly Bemisia tabaci in the phloem of tomato plants confers resistance to tomato yellow leaf curl virus.

Some (perhaps all) plant viruses transmitted in a circulative manner by their insect vectors avoid destruction in the haemolymph by interacting with GroEL homologues, ensuring transmission. We have previously shown that the phloem-limited begomovirus tomato yellow leaf curl virus (TYLCV) interacts in vivo and in vitro with GroEL produced by the whitefly vector Bemisia tabaci. In this study, we have exploited this phenomenon to generate transgenic tomato plants expressing the whitefly GroEL in their phloem. We postulated that following inoculation, TYLCV particles will be trapped by GroEL in the plant phloem, thereby inhibiting virus replication and movement, thereby rendering the plants resistant. A whitefly GroEL gene was cloned in an Agrobacterium vector under the control of an Arabidopsis phloem-specific promoter, which was used to transform two tomato genotypes. During three consecutive generations, plants expressing GroEL exhibited mild or no disease symptoms upon whitefly-mediated inoculation of TYLCV. In vitro assays indicated that the sap of resistant plants contained GroEL-TYLCV complexes. Infected resistant plants served as virus source for whitefly-mediated transmission as effectively as infected non-transgenic tomato. Non-transgenic susceptible tomato plants grafted on resistant GroEL-transgenic scions remained susceptible, although GroEL translocated into the grafted plant and GroEL-TYLCV complexes were detected in the grafted tissues.

Nuclear DNA content of the whitefly Bemisia tabaci (Aleyrodidae: Hemiptera) estimated by flow cytometry.

The nuclear DNA content of the whitefly Bemisia tabaci (Gennnadius) was estimated using flow cytometry. Male and female nuclei were stained with propidium iodide and their DNA content was estimated using chicken red blood cells and Arabidopsis thaliana L. (Brassicaceae) as external standards. The estimated nuclear DNA content of male and female B. tabaci was 1.04 and 2.06 pg, respectively. These results corroborated previous reports based on chromosome counting, which showed that B. tabaci males are haploid and females are diploid. Conversion between DNA content and genome size (1 pg DNA=980 Mbp) indicate that the haploid genome size of B. tabaci is 1020 Mbp, which is approximately five times the size of the genome of the fruitfly Drosophila melanogaster Meigen. These results provide an important baseline that will facilitate genomics-based research for the B. tabaci complex.

Whiteflies (Bemisia tabaci) issued from eggs bombarded with infectious DNA clones of Tomato yellow leaf curl virus from Israel (TYLCV) are able to infect tomato plants.

We have reported previously that Tomato yellow leaf curl virus from Israel (TYLCV) penetrates the reproductive system of its vector, the whitefly Bemisia tabaci biotype B, and may be transmitted to progeny. In order to mimic this phenomenon and to understand how TYLCV accompanies the development of the insect, we have bombarded B. tabaci eggs with an infectious DNA clone of TYLCV. After a linear full-length genomic copy of TYLCV DNA was delivered to eggs, the DpnI-sensitive DNA became circular and DpnI resistant. When a dimeric copy of TYLCV DNA was delivered to eggs, the viral DNA was detected in all the whitefly developmental stages. Adult insects that developed from the treated eggs were able to infect tomato test plants with variable frequency. Viral DNA was detected in the progeny of whiteflies that developed from eggs bombarded with TYLCV. Similarly, when insect eggs were bombarded with a dimeric copy of an infectious clone of the genome of Tomato yellow leaf curl virus from Sardinia, Italy (TYLCSV), adults that eclosed from the treated eggs were able to infect tomato test plants.