Human cytomegalovirus has a complex double-stranded DNA genome of Ϸ240,000 bp that contains Ϸ150 ORFs likely to encode proteins, most of whose functions are not well understood. We have used an infectious bacterial artificial chromosome to introduce 413 defined insertion and substitution mutations into the human cytomegalovirus AD169 genome by random and sitedirected transposon mutagenesis. Mutations were produced in all unique ORFs with a high probability of encoding proteins for which mutants have not been previously documented and in many previously characterized ORFs. The growth of selected mutants was assayed in cultured human fibroblasts, and we now recognize 41 essential, 88 nonessential, and 27 augmenting ORFs. Most essential and augmenting genes are located in the central region, and nonessential genes generally cluster near the ends of the viral genome. Human cytomegalovirus (HCMV) is the prototypic ␤-herpes virus and a ubiquitous human pathogen. Although infections in healthy children and adults are generally asymptomatic, HCMV is a leading viral cause of birth defects and a major cause of morbidity and mortality in immunocompromised individuals (1).HCMV contains a complex double-stranded DNA genome of Ϸ240,000 bp, the largest genome for a virus known to infect humans. The laboratory strain of HCMV, AD169, contains Ϸ150 ORFs likely to encode proteins (2-5). Most ORFs have not been well studied due to the limited host range and slow growth of HCMV in cultured cells and the lack of efficient tools to generate mutant viruses. Recently, the HCMV genome has been cloned as an infectious bacterial artificial chromosome (BAC) (6-9), greatly facilitating its genetic manipulation (8, 10).We previously described an infectious BAC clone of HCMV AD169, termed pAD͞Cre (6). The BAC vector is flanked by LoxP sites and contains a Cre-recombinase gene that is modified by the insertion of an intron into its coding sequence. Consequently, Cre is not expressed in bacterial cells, but it is expressed when its transcript is spliced in human cells and the BAC vector is excised from the virus. Now we report the use of both random and site-directed transposon mutagenesis to introduce 413 defined insertion and substitution mutations into the HCMV AD169 genome residing in pAD͞Cre. Mutations were produced in all ORFs with a high probability of encoding proteins for which mutants have not been previously documented and in many previously characterized ORFs. We have begun to systematically delineate the functions of viral ORFs in HCMVinfected cells by analyzing the growth of HCMV mutants in cultured human fibroblasts. We now recognize 41 essential, 88 nonessential, and 27 augmenting ORFs. This work describes a functional map of the complete HCMV genome and provides a foundation for future genetic studies. MethodsCells, Viruses, and Plasmids. Primary human foreskin fibroblasts at passage 8-15 were propagated in medium supplemented with 10% FCS. The HCMV strain AD169 BAC, pAD͞Cre (6), was the wild-type parent of all mutant viruses. A...

Although exhibiting limited genetic polymorphism, the very large genome of H. vastatrix (c. 797 Mbp) conceals great pathological diversity, with more than 50 physiological races. Gene expression studies have revealed a very precocious activation of signalling pathways and production of putative effectors, suggesting that the plant-fungus dialogue starts as early as at the germ tube stage, and have provided clues for the identification of avr genes.

The human cytomegalovirus UL99-encoded pp28 is a myristylated phosphoprotein that is a constituent of the virion. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. In the infected cell, pp28 is found in a cytoplasmic compartment derived from the Golgi apparatus, where the virus buds into vesicles to acquire its final membrane. We have constructed two mutants of human cytomegalovirus that fail to produce the pp28 protein, a substitution mutant (BADsubUL99) and a point mutant (BADpmUL99), and we have propagated them by complementation in pp28-expressing fibroblasts. Both mutant viruses are profoundly defective for growth in normal fibroblasts; no infectious virus could be detected after infection. Whereas normal levels of viral DNA and late proteins were observed in mutant virus-infected cells, large numbers of tegument-associated capsids accumulated in the cytoplasm that failed to acquire an envelope. We conclude that pp28 is required for the final envelopment of the human cytomegalovirus virion in the cytoplasm.Human cytomegalovirus (HCMV) is the prototypical member of the betaherpesvirus family. Seroepidemiologic studies have shown that HCMV infection is widespread in the human population in both industrial and developing regions. In healthy individuals infection is generally asymptomatic, but the virus can cause serious disease in people with immature or compromised immune systems. It is the leading infectious disease cause of birth defects and a life-threatening adventitious agent in transplant recipients and AIDS patients (40,43).In virions, the double-stranded HCMV DNA resides within a capsid that is surrounded first by a tegument layer and then by an envelope. The tegument domain, which is unique to herpesvirus particles, contains approximately 30 virus-encoded proteins (1,5,14). Since they are components of virions, tegument proteins are delivered to cells at the very start of infection and they have the potential to function even before the viral genome is activated. For example, the UL83-encoded pp65 protein has been reported to block major histocompatibility complex class I presentation of a viral immediate-early protein (21), the UL47 protein acts during disassembly of the newly infecting virus particle (8), the UL82-encoded pp71 protein is a transcriptional activator (32) that helps to activate the immediate-early genes within infected cells (10), and the UL69 protein blocks cell cycle progression (23).The pp28 protein of HCMV (31, 36) is a 190-amino-acid myristylated (51) phosphoprotein (39) that is expressed as a true late protein (28, 37), i.e., it is synthesized only after the onset of viral DNA replication. The pp28 protein is encoded by UL99, the last open reading frame positioned within a family of 3Ј-coterminal transcripts that share the same polyadenylation site (61) (Fig. 1A). It is one of the most abundant constituents of the tegument layer (5, 31) and is highly immunogenic (30,39,44).Whereas some teg...

Considerable success has been obtained in the use of classical breeding to control economically important plant diseases, such as the coffee leaf rust and the coffee berry disease (CBD). There is a strong consensus that growing genetically resistant varieties is the most appropriate cost effective means of managing plant diseases and is one of the key components of crop improvement. It has also been recognized that a better knowledge of both, the pathogens and the plant defence mechanisms will allow the development of novel approaches to enhance the durability of resistance. After a brief description of concepts in the field of plant disease resistance, we attempt to give a view of the research progress on coffee leaf rust and CBD concerned with the pathogens infection and variability, coffee breeding for resistance and coffee resistance mechanisms. Key words: Coffea, Colletotrichum kahawae, Hemileia vastatrix, coffee breeding, resistance.Resistência do cafeeiro para suas principais doenças: ferrugem alaranjada das folhas e antracnose dos frutos: Sucesso considerável tem sido obtido no uso do melhoramento clássico para o controle de doenças de plantas economicamente importantes, tais como a ferrugem alaranjada das folhas e a antracnose dos frutos do cafeeiro (CBD). Há um grande consenso de que o uso de plantas geneticamente resistentes é o meio mais apropriado e eficaz em termos de custos do controle das doenças das plantas, sendo também um dos elementos chave do melhoramento da produção agrícola. Tem sido também reconhecido que um melhor conhecimento do agente patogênico e dos mecanismos de defesa das plantas permitirá o desenvolvimento de novas abordagens no sentido de aumentar a durabilidade da resistência. Após uma breve descrição de conceitos na área da resistência das plantas às doenças, nesta revisão tentou-se dar uma idéia do progresso na investigação da ferrugem alaranjada do cafeeiro e do CBD relativamente ao processo de infecção e variabilidade dos agentes patogênicos, melhoramento do cafeeiro para a resistência e mecanismos de resistência do cafeeiro. Palavras-chave: Coffea, Colletotrichum kahawae, Hemileia vastatrix, melhoramento do cafeeiro, resistência.

Coffee (Coffea arabica L.), one of the key export and cash crops in tropical and subtropical countries, suffers severe losses from the rust fungus Hemileia vastatrix. The transcriptome of H. vastatrix was analysed during a compatible interaction with coffee to obtain an exhaustive repertoire of the genes expressed during infection and to identify potential effector genes. Large-scale sequencing (454-GS-FLEX Titanium) of mixed coffee and rust cDNAs obtained from 21-day rust-infected leaves generated 352 146 sequences which assembled into 22 774 contigs. In the absence of any reference genomic sequences for Coffea or Hemileia, specific trinucleotide frequencies within expressed sequence tags (ESTs) and blast homology against a set of dicots and basidiomycete genomes were used to distinguish pathogen from plant sequences. About 30% (6763) of the contigs were assigned to H. vastatrix and 61% (13 951) to C. arabica. The majority (60%) of the rust sequences did not show homology to any genomic database, indicating that they were potential novel fungal genes. In silico analyses of the 6763 H. vastatrix contigs predicted 382 secreted proteins and identified homologues of the flax rust haustorially expressed secreted proteins (HESPs) and bean rust transferred protein 1 (RTP1). These rust candidate effectors showed conserved amino-acid domains and conserved patterns of cysteine positions suggestive of conserved functions during infection of host plants. Quantitative reverse transcription-polymerase chain reaction profiling of selected rust genes revealed dynamic expression patterns during the time course of infection of coffee leaves. This study provides the first valuable genomic resource for the agriculturally important plant pathogen H. vastatrix and the first comprehensive C. arabica EST dataset.