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...

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...

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.

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.

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Rust fungi (Basidiomycota, Pucciniales) are biotrophic plant pathogens which exhibit diverse complexities in their life cycles and host ranges. The completion of genome sequencing of a few rust fungi has revealed the occurrence of large genomes. Sequencing efforts for other rust fungi have been hampered by uncertainty concerning their genome sizes. Flow cytometry was recently applied to estimate the genome size of a few rust fungi, and confirmed the occurrence of large genomes in this order (averaging 225.3 Mbp, while the average for Basidiomycota was 49.9 Mbp and was 37.7 Mbp for all fungi). In this work, we have used an innovative and simple approach to simultaneously isolate nuclei from the rust and its host plant in order to estimate the genome size of 30 rust species by flow cytometry. Genome sizes varied over 10-fold, from 70 to 893 Mbp, with an average genome size value of 380.2 Mbp. Compared to the genome sizes of over 1800 fungi, Gymnosporangium confusum possesses the largest fungal genome ever reported (893.2 Mbp). Moreover, even the smallest rust genome determined in this study is larger than the vast majority of fungal genomes (94%). The average genome size of the Pucciniales is now of 305.5 Mbp, while the average Basidiomycota genome size has shifted to 70.4 Mbp and the average for all fungi reached 44.2 Mbp. Despite the fact that no correlation could be drawn between the genome sizes, the phylogenomics or the life cycle of rust fungi, it is interesting to note that rusts with Fabaceae hosts present genomes clearly larger than those with Poaceae hosts. Although this study comprises only a small fraction of the more than 7000 rust species described, it seems already evident that the Pucciniales represent a group where genome size expansion could be a common characteristic. This is in sharp contrast to sister taxa, placing this order in a relevant position in fungal genomics research.

Anthracnose is an important disease affecting mature olive fruits, causing significant yield losses, and poor fruit and oil quality. In Portugal, high anthracnose incidence was recorded during 2003-2007 with 41% of 908 orchards surveyed displaying disease symptoms. In another 14% of the orchards, the pathogen was recorded in symptomless plants. Disease severity was on average 36%, frequently reaching 100%. In Portugal, anthracnose is endemic to neglected orchards of susceptible cultivars, but under favourable conditions it can also severely affect less susceptible cultivars. Pathogens were genetically heterogeneous, with Colletotrichum acutatum genetic group A2 as the most frequent (80%), followed by group A4 (12%) and group A5 along with C. gloeosporioides (3-4%), while groups A3 and A6 of C. acutatum were sporadic. Important geographic variations were observed in the frequencies of these populations, accompanied by year-to-year populational shifts. Epidemiology and histopathology studies showed the presence of the pathogens on vegetative organs year-round, particularly on olive leaves and branches, and on weeds. These represent inoculum reservoirs where secondary conidiation occurs, and conidia are then dispersed by spring rains reaching flowers and young fruits or by autumn rains reaching pre-mature fruits. Unripe fruits were colonized without showing symptoms up to penetration of the cuticle, but further colonization and symptom production was completed only as fruits matured. These findings challenge current control practices, particularly the timing of fungicide treatment, and contribute to improved disease management.