First report of the physiological race (XXIV) of Hemileia vastatrix (Coffee Leaf Rust) in Hawaii

This research was funded by the Foundation for Science and Technology (FCT) UNIT (UID/AGR/04129/2020) of LEAF- -Linking Landscape, Environment, Agriculture and Food, Research Unit.info:eu-repo/semantics/publishedVersio

A chromosome-level genome resource for studying virulence mechanisms and evolution of the coffee rust pathogen Hemileia vastatrix

Recurrent epidemics of coffee leaf rust, caused by the fungal pathogen Hemileia vastatrix, have constrained the sustainable production of Arabica coffee for over 150 years. The ability of H. vastatrix to overcome resistance in coffee cultivars and evolve new races is inexplicable for a pathogen that supposedly only utilizes clonal reproduction. Understanding the evolutionary complexity between H. vastatrix and its only known host, including determining how the pathogen evolves virulence so rapidly is crucial for disease management. Achieving such goals relies on the availability of a comprehensive and high-quality genome reference assembly. To date, two reference genomes have been assembled and published for H. vastatrix that, while useful, remain fragmented and do not represent chromosomal scaffolds. Here, we present a complete scaffolded pseudochromosome-level genome resource for H. vastatrix strain 178a (Hv178a). Our initial assembly revealed an unusually high degree of gene duplication (over 50% BUSCO basidiomycota_odb10 genes). Upon inspection, this was predominantly due to a single scaffold that itself showed 91.9% BUSCO Completeness. Taxonomic analysis of predicted BUSCO genes placed this scaffold in Exobasidiomycetes and suggests it is a distinct genome, which we have named Hv178a associated fungal genome (Hv178a AFG). The high depth of coverage and close association with Hv178a raises the prospect of symbiosis, although we cannot completely rule out contamination at this time. The main Ca. 546 Mbp Hv178a genome was primarily (97.7%) localised to 11 pseudochromosomes (51.5 Mb N50), building the foundation for future advanced studies of genome structure and organization.info: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

The story of coffee: legend and truth

FCT and FEDER funds refs. PTDC/ASP-PLA/32429/ 2017 (POCI-01-0145-FEDER-032429) and PTDC/ ASP-PLA/29779/2017 and by FCT UNIT (UID/AGR/ 04129/2020) of LEAF (Linking Landscape, Environment, Agriculture and Food) Research Unit.info:eu-repo/semantics/publishedVersio

Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae.

Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica-C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the model-based method used by NormFinder software. For C. arabica-C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction

Prediction of the optimal number of reference genes required for effective normalization.

<p>Pairwise variation (V) of the candidate reference genes calculated by geNorm using the two different datasets studied: i) all <i>C</i>. <i>arabica-C</i>. <i>kahawae</i> samples; ii) all <i>C</i>. <i>kahawae</i> samples.</p

Detailed description of candidate reference genes, genes of interest, primer sets and qPCR amplification conditions.

<p>Detailed description of candidate reference genes, genes of interest, primer sets and qPCR amplification conditions.</p

Details on <i>Colletotrichum kahawae</i> isolates regarding its geographical origin and aggressiveness pattern in <i>Coffea arabica</i> (var. Caturra).

<p>Details on <i>Colletotrichum kahawae</i> isolates regarding its geographical origin and aggressiveness pattern in <i>Coffea arabica</i> (var. Caturra).</p

Relative quantification of <i>cat2</i> expression using the best and the Worst normalization factors (NF).

<p>Expression profiles are presented per isolate (Ang29 (A), Zim 12 (B), Que2 (C) and Ang67 (D)), during the early stages of infection process and growth (Ap: Appressoria; M: Mycelium). The <i>C</i>. <i>arabica–C</i>. <i>kahawae</i> samples were normalized with <b>NF Global</b> <i>C</i>. <i>arabica—C</i>. <i>kahawae</i> interaction (<i>PP1; Act; ck34620)</i> and <b>NF Worst</b> (<i>ck20430; ck48742; ck36020)</i>, while the <i>C</i>. <i>kahawae</i> samples were normalized with <b>NF Global</b> <i>C</i>. <i>Kahawae</i> (<i>PP1; Act; ck20430</i>) and <b>NF Worst</b> (<i>ck34620; ck36020</i>).</p