Evaluation of virus-induced gene silencing (VIGS) in coffee plants

29th Conference of Association for the Science and Information on Coffee, 11 Sept. - 14 Sept. 2023 Hanoi, Vietnaminfo:eu-repo/semantics/publishedVersio

Exploring the role of sugars in the Kawisari coffee resistance to Hemileia vastatrix

29th Conference of Association for the Science and Information on Coffee, 11 Sept. - 14 Sept. 2023 Hanoi, Vietnaminfo:eu-repo/semantics/publishedVersio

Integrative transcriptomic and metabolomic approaches to unravel the resistance profile of Kawisari coffee against Hemileia vastatrix

29th Conference of Association for the Science and Information on Coffee, 11 Sept. - 14 Sept. 2023 Hanoi, Vietnaminfo:eu-repo/semantics/publishedVersio

Characterization of the transcriptional signatures associated with resistance and susceptibility to Hemileia vastatrix in the Kawisari coffee hybrid

Coffee leaf rust (CLR), a disease caused by the biotrophic fungus Hemileia vastatrix (Hv), is the main threat to the worldwide production of Arabica coffee. The gradual breakdown of resistance in coffee varieties in the last years has highlighted the need for novel sources of resistance to CLR. This work aimed to unveil the cellular and molecular resistance profile of the Kawisari hybrid (C. arabica x C. liberica), a genotype used as a resistance donor in Arabica breeding programs in India. This coffee genotype was inoculated with two Hv races that triggered either resistance or susceptibility. Progress of infection was monitored using light microscopy. Simultaneously, we conducted a time-course RNA-seq characterization of the transcriptional responses. The microscopic studies showed that the post-haustorial resistance of Kawisari was associated with the hypersensitive response, accumulation of phenolic-like compounds and haustorium encasement with callose. The transcriptomic analysis suggest the downregulation of host primary metabolism genes at the early onset of infection, followed later by activation of genes functionally associated with multiple plant defense responses, including salicylic acid and jasmonate hormonal signaling. Resistance was also accompanied by the differential regulation of genes associated with phenylpropanoid metabolism and lignin biosynthesis. Our results, further validated by qPCR, provide important new insight into the molecular mechanisms underpinning resistance against CLR in this coffee genotype.Foundation for Science and Technology (FCT) and FEDER funds through PORNorte under the project CoffeeRES PTDC/ASPPLA/ 29779/2017 and by FCT UNIT LEAF (UID/AGR/04129/2020).info:eu-repo/semantics/publishedVersio

Genome size analyses of Pucciniales reveal the largest fungal genomes

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 151.5 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 1,800 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 7,000 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

Expression profiling of genes involved in the biotrophic colonisation of [i]Coffea arabica[/i] leaves by [i]Hemileia vastatrix[/i]

Coffee Leaf Rust, caused by Hemileia vastatrix, is the most important disease of Arabica coffee (Coffea arabica), which prompts studies aimed at understanding the genetic basis of this pathogen as well as its complex interaction with the host. In this work, 11 genes, putatively involved in signalling, establishment and maintenance of biotrophy (transport and metabolism), were characterised, and their expression profiles during host infection were assessed by RT-qPCR in three compatible coffee-rust interactions comprising two different rust races. The profiles of two chitin deacetylases (CD) and a heterotrimeric G-protein alpha subunit transcripts suggest that these enzymes are involved in host-pathogen recognition and establishment of biotrophy at early stages of infection, and the late expression of the CD1 gene was also recorded. Different expression profiles were observed for a MAP kinase gene between the two rust races, suggesting that this gene may be involved in the differentiation of infection structures in a race-specific pattern. Two amino acid transporters, an invertase, a hexose transporter and a mannitol dehydrogenase presented expression profiles similar to those reported in other rust fungi, indicating a fairly conserved genetic programme related to host infection in rust fungi. The strong upregulation of a Uromyces fabae rust transferred protein 1 orthologous gene was observed in H. vastatrix in planta structures, suggesting that this gene may also play a role during the establishment and the maintenance of biotrophy in coffee leaves. Overall, our results provide valuable insights to the current understanding of the biotrophic interaction between H. vastatrix - C. arabica at the molecular level and will contribute to a reasoned and sustainable use of resistant genotypes

Phylogenetic analysis of Hemileia vastatrix and related taxa using a genome-scale approach

For more than a century, Coffee Leaf Rust caused by the biotrophic fungus Hemileia vastatrix, has increasingly stood out as one of the major factors hampering Arabica coffee production. Since its first historical outburst in the 19th century in Sri Lanka, this disease has rapidly spread worldwide and currently occurs in nearly all the regions of the world where coffee is grown. Despite its widespread distribution and negative economic impact, little is known about its evolutionary origin and phylogenetic placement in the fungal tree of life. Attaining this knowledge, however, would provide fundamental insights on the evolutionary context in which H. vastatrix emerged, which in turn could have important implications to understand the evolution of pathogenicity in this pathogen and in other rusts as well. With this in mind, we are undertaking a genomescale approach that will allow H. vastatrix and other rust fungi to be placed in the fungal tree of life with unprecedented detail by using complete proteomes of five Pucciniomycotina species, approximately 250 000 publicly available EST sequences from several Pucciniomycotina species and H. vastatrix’s recently obtained transcriptome. A high quality matrix that includes orthologs, co-orthologs and recent paralogs is currently being prepared with a sophisticated orthology detection strategy. On a first approach, we have been able to indentify at least 1040 single-copy orthologs that will be used for the phylogenetic analyses. Providing robust and resolved phylogenetic relationships will lay the ground for the identification of genes or gene families that are exclusive to H. vastatrix as well as genes of rapid evolution and/or subject of positive selection, which would be prime targets for functional studies aiming at disease control and prevention. This information will ultimately contribute significantly to advance our knowledge on H. vastatrix’s pathogenicity

Coffee Leaf Rust Resistance: An Overview

Coffee is one of the most important cash crops and beverages. Several diseases caused by fungi, bacteria, and viruses can affect coffee plantations and compromise production. Coffee leaf rust (CLR), caused by the biotrophic fungus Hemileia vastatrix is the top fungal disease, representing a permanent threat to sustainable Arabica coffee production for more than a century. This review provides a comprehensive survey of the most common coffee diseases, their importance, and geographic distribution, with an emphasis on coffee leaf rust. Summing up the progress obtained so far from different research fields on the coffee–H. vastatrix interaction, we revisited the pathogen genetic diversity and population dynamics, and the complex mechanisms underlying plant resistance/immunity. We also highlight how new advanced technologies can provide avenues for a deeper understanding of this pathosystem, which is crucial for devising more reliable and long-term strategies for disease control.info:eu-repo/semantics/publishedVersio

Overview of the functional virulent genome of the coffee leaf rust pathogen Hemileia vastatrix

International audienceCoffee plants are seriously affected by leaf rust (Hemileia vastatrix) and loss of resistance to emerging races is a current threat. A deep knowledge of the mechanisms of pathogenicity is necessary for better understanding the plant resistance mechanisms, particularly because the Coffea spp. – H. vastatrix interaction follows the gene for gene theory, enabling the inference of virulence genes in the pathogen and the identification of susceptibility genes in the plant from resistance/susceptibility phenotypes. In the absence of H. vastatrix genome sequence information, transcriptomic analysis is the most effective gene discovery strategy, prompting gene family identification, the establishment of phylogenetic relationships and the development of PCR based molecular markers. High-throughput sequencing of cDNA transcripts by 454 pyrosequencing is capable of producing millions of bases per run, enabling large-scale expression analysis. The objective of this work is the characterisation and comparison of H. vastratrix transcriptomes at three key differentiation/infection stages (germinated uredospores, appressoria and intercellular hyphae with haustoria) for an isolate from race XIV (containing the virulence genes v2,3,4,5). For such, cDNA was obtained from germinated uredospores and appressoria produced in vitro, as well as from intercellular hyphae with haustoria (infected coffee leaves 21 days after inoculation) and subjected to GS-Flex Titanium 454 sequencing (each library comprising over 500000 reads of 300-600bp). Prior to annotation, for the in vivo sample and in the absence of genome sequencing information for both organisms, H. vastatrix transcripts were separated from Coffea sp. transcripts by the analysis of codon usage in ESTs (programme EST3), of GC content, and of blastn homology score against angiosperms and basidiomycetes genomes, as well as of blastx homology score against coffee and Pucciniales EST libraries. The three H. vastatrix EST collections (germinating uredospores, appressoria and intercellular hyphae with haustoria) were annotated using international databases (nr, Swissprot, GO, KOG), aiming to attribute a putative gene function. A particular attention was given to the comparison with the Pucciniales genome sequences (Melampsora larici-populina and Puccinia spp.) and Pucciniales EST databases, in order to identify homologues of genes with relevant function in pathogenicity. The libraries representing the three H. vastatrix differentiation/infection stages were compared among them, in order to identify genes differentially expressed. Genes identified were further studied by RT-qPCR expression analysis in a detailed time-course of H. vastatrix differentiation/infection (Vieira et al., 2010). The identification of H. vastatrix genes involved in pathogenicity will lead to a better understanding of the molecular basis of coffee rust gene for gene interaction