Quercus leaf extracts display curative effects against Candidatus Liberibacter asiaticus that restore leaf physiological parameters in HLB-affected citrus trees.

Citrus greening, also called Huanglongbing (HLB), is one of the most destructive citrus diseases worldwide. It is caused by the fastidious gram-negative α-proteobacteria bacterium Candidatus Liberibacter asiaticus (CLas) and vectored by the Asian citrus psyllid (ACP), Diaphorina citri. Currently, there is no cure for HLB, no compounds have been successful in controlling HLB, and no sustainable management practices have been established for the disease. Thus, searching for alternative citrus greening disease mitigation strategies is considered an urgent priority for a sustainable citrus industry. The aim of this study was to use compounds extracted from oak, Quercus hemisphaerica, and to assess the antibacterial effects of these against CLas-infected citrus plants. The application of aqueous oak leaf extracts showed substantial inhibitory effects against CLas in citrus plants and the activity of genes related to starch. Significant differences were also observed in plant phenotypic and physiological traits after treatments. Citrus plants treated with oak extracts displayed an increase in stomatal conductance, chlorophyll content and nutrient uptake concurrently with a reduction of CLas titer, when compared to citrus plants treated with just water. The information provided from this study suggests a new management treatment program to effectively deal with the HLB disease.

Gene expression polymorphism underpins evasion of host immunity in an asexual lineage of the Irish potato famine pathogen.

BACKGROUND: Outbreaks caused by asexual lineages of fungal and oomycete pathogens are a continuing threat to crops, wild animals and natural ecosystems (Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ, Nature 484:186-194, 2012; Kupferschmidt K, Science 337:636-638, 2012). However, the mechanisms underlying genome evolution and phenotypic plasticity in asexual eukaryotic microbes remain poorly understood (Seidl MF, Thomma BP, BioEssays 36:335-345, 2014). Ever since the 19th century Irish famine, the oomycete Phytophthora infestans has caused recurrent outbreaks on potato and tomato crops that have been primarily caused by the successive rise and migration of pandemic asexual lineages (Goodwin SB, Cohen BA, Fry WE, Proc Natl Acad Sci USA 91:11591-11595, 1994; Yoshida K, Burbano HA, Krause J, Thines M, Weigel D, Kamoun S, PLoS Pathog 10:e1004028, 2014; Yoshida K, Schuenemann VJ, Cano LM, Pais M, Mishra B, Sharma R, Lanz C, Martin FN, Kamoun S, Krause J, et al. eLife 2:e00731, 2013; Cooke DEL, Cano LM, Raffaele S, Bain RA, Cooke LR, Etherington GJ, Deahl KL, Farrer RA, Gilroy EM, Goss EM, et al. PLoS Pathog 8:e1002940, 2012). However, the dynamics of genome evolution within these clonal lineages have not been determined. The objective of this study was to use a comparative genomics and transcriptomics approach to determine the molecular mechanisms that underpin phenotypic variation within a clonal lineage of P. infestans. RESULTS: Here, we reveal patterns of genomic and gene expression variation within a P. infestans asexual lineage by comparing strains belonging to the South American EC-1 clone that has dominated Andean populations since the 1990s (Yoshida K, Burbano HA, Krause J, Thines M, Weigel D, Kamoun S, PLoS Pathog 10e1004028, 2014; Yoshida K, Schuenemann VJ, Cano LM, Pais M, Mishra B, Sharma R, Lanz C, Martin FN, Kamoun S, Krause J, et al. eLife 2:e00731, 2013; Delgado RA, Monteros-Altamirano AR, Li Y, Visser RGF, van der Lee TAJ, Vosman B, Plant Pathol 62:1081-1088, 2013; Forbes GA, Escobar XC, Ayala CC, Revelo J, Ordonez ME, Fry BA, Doucett K, Fry WE, Phytopathology 87:375-380, 1997; Oyarzun PJ, Pozo A, Ordonez ME, Doucett K, Forbes GA, Phytopathology 88:265-271, 1998). We detected numerous examples of structural variation, nucleotide polymorphisms and loss of heterozygosity within the EC-1 clone. Remarkably, 17 genes are not expressed in one of the two EC-1 isolates despite apparent absence of sequence polymorphisms. Among these, silencing of an effector gene was associated with evasion of disease resistance conferred by a potato immune receptor. CONCLUSIONS: Our findings highlight the molecular changes underpinning the exceptional genetic and phenotypic plasticity associated with host adaptation in a pandemic clonal lineage of a eukaryotic plant pathogen. We observed that the asexual P. infestans lineage EC-1 can exhibit phenotypic plasticity in the absence of apparent genetic mutations resulting in virulence on a potato carrying the Rpi-vnt1.1 gene. Such variant alleles may be epialleles that arose through epigenetic changes in the underlying genes.

Arabidopsis late blight: infection of a nonhost plant by Albugo laibachii enables full colonization by Phytophthora infestans.

The oomycete pathogen Phytophthora infestans causes potato late blight, and as a potato and tomato specialist pathogen, is seemingly poorly adapted to infect plants outside the Solanaceae. Here, we report the unexpected finding that P. infestans can infect Arabidopsis thaliana when another oomycete pathogen, Albugo laibachii, has colonized the host plant. The behaviour and speed of P. infestans infection in Arabidopsis pre-infected with A. laibachii resemble P. infestans infection of susceptible potato plants. Transcriptional profiling of P. infestans genes during infection revealed a significant overlap in the sets of secreted-protein genes that are induced in P. infestans upon colonization of potato and susceptible Arabidopsis, suggesting major similarities in P. infestans gene expression dynamics on the two plant species. Furthermore, we found haustoria of A. laibachii and P. infestans within the same Arabidopsis cells. This Arabidopsis-A. laibachii-P. infestans tripartite interaction opens up various possibilities to dissect the molecular mechanisms of P. infestans infection and the processes occurring in co-infected Arabidopsis cells.

A Recent Expansion of the RXLR Effector Gene Avrblb2 Is Maintained in Global Populations of Phytophthora infestans Indicating Different Contributions to Virulence.

The introgression of disease resistance (R) genes encoding immunoreceptors with broad-spectrum recognition into cultivated potato appears to be the most promising approach to achieve sustainable management of late blight caused by the oomycete pathogen Phytophthora infestans. Rpi-blb2 from Solanum bulbocastanum shows great potential for use in agriculture based on preliminary potato disease trials. Rpi-blb2 confers immunity by recognizing the P. infestans avirulence effector protein AVRblb2 after it is translocated inside the plant cell. This effector belongs to the RXLR class of effectors and is under strong positive selection. Structure-function analyses revealed a key polymorphic amino acid (position 69) in AVRblb2 effector that is critical for activation of Rpi-blb2. In this study, we reconstructed the evolutionary history of the Avrblb2 gene family and further characterized its genetic structure in worldwide populations. Our data indicate that Avrblb2 evolved as a single-copy gene in a putative ancestral species of P. infestans and has recently expanded in the Phytophthora spp. that infect solanaceous hosts. As a consequence, at least four variants of AVRblb2 arose in P. infestans. One of these variants, with a Phe residue at position 69, evades recognition by the cognate resistance gene. Surprisingly, all Avrblb2 variants are maintained in pathogen populations. This suggests a potential benefit for the pathogen in preserving duplicated versions of AVRblb2, possibly because the variants may have different contributions to pathogen fitness in a diversified solanaceous host environment.

Effector specialization in a lineage of the Irish potato famine pathogen.

Accelerated gene evolution is a hallmark of pathogen adaptation following a host jump. Here, we describe the biochemical basis of adaptation and specialization of a plant pathogen effector after its colonization of a new host. Orthologous protease inhibitor effectors from the Irish potato famine pathogen, Phytophthora infestans, and its sister species, Phytophthora mirabilis, which is responsible for infection of Mirabilis jalapa, are adapted to protease targets unique to their respective host plants. Amino acid polymorphisms in both the inhibitors and their target proteases underpin this biochemical specialization. Our results link effector specialization to diversification and speciation of this plant pathogen.

The rise and fall of the Phytophthora infestans lineage that triggered the Irish potato famine.

Phytophthora infestans, the cause of potato late blight, is infamous for having triggered the Irish Great Famine in the 1840s. Until the late 1970s, P. infestans diversity outside of its Mexican center of origin was low, and one scenario held that a single strain, US-1, had dominated the global population for 150 years; this was later challenged based on DNA analysis of historical herbarium specimens. We have compared the genomes of 11 herbarium and 15 modern strains. We conclude that the 19th century epidemic was caused by a unique genotype, HERB-1, that persisted for over 50 years. HERB-1 is distinct from all examined modern strains, but it is a close relative of US-1, which replaced it outside of Mexico in the 20th century. We propose that HERB-1 and US-1 emerged from a metapopulation that was established in the early 1800s outside of the species' center of diversity. DOI:http://dx.doi.org/10.7554/eLife.00731.001.

Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen.

Pest and pathogen losses jeopardise global food security and ever since the 19(th) century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.

Qualitative and quantitative late blight resistance in the potato cultivar Sarpo Mira is determined by the perception of five distinct RXLR effectors.

Potato defends against Phytophthora infestans infection by resistance (R)-gene-based qualitative resistance as well as a quantitative field resistance. R genes are renowned to be rapidly overcome by this oomycete, and potato cultivars with a decent and durable resistance to current P. infestans populations are hardly available. However, potato cultivar Sarpo Mira has retained resistance in the field over several years. We dissected the resistance of 'Sarpo Mira' in a segregating population by matching the responses to P. infestans RXLR effectors with race-specific resistance to differential strains. The resistance is based on the combination of four pyramided qualitative R genes and a quantitative R gene that was associated with field resistance. The qualitative R genes include R3a, R3b, R4, and the newly identified Rpi-Smira1. The qualitative resistances matched responses to avirulence (AVR)3a, AVR3b, AVR4, and AVRSmira1 RXLR effectors and were overcome by particular P. infestans strains. The quantitative resistance was determined to be conferred by a novel gene, Rpi-Smira2. It was only detected under field conditions and was associated with responses to the RXLR effector AvrSmira2. We foresee that effector-based resistance breeding will facilitate selecting and combining qualitative and quantitative resistances that may lead to a more durable resistance to late blight.

Understanding and exploiting late blight resistance in the age of effectors.

Potato (Solanum tuberosum) is the world's third-largest food crop. It severely suffers from late blight, a devastating disease caused by Phytophthora infestans. This oomycete pathogen secretes host-translocated RXLR effectors that include avirulence (AVR) proteins, which are targeted by resistance (R) proteins from wild Solanum species. Most Solanum R genes appear to have coevolved with P. infestans at its center of origin in central Mexico. Various R and Avr genes were recently cloned, and here we catalog characterized R-AVR pairs. We describe the mechanisms that P. infestans employs for evading R protein recognition and discuss partial resistance and partial virulence phenotypes in the context of our knowledge of effector diversity and activity. Genome-wide catalogs of P. infestans effectors are available, enabling effectoromics approaches that accelerate R gene cloning and specificity profiling. Engineering R genes with expanded pathogen recognition has also become possible. Importantly, monitoring effector allelic diversity in pathogen populations can assist in R gene deployment in agriculture.

Presence/absence, differential expression and sequence polymorphisms between PiAVR2 and PiAVR2-like in Phytophthora infestans determine virulence on R2 plants.

• A detailed molecular understanding of how oomycete plant pathogens evade disease resistance is essential to inform the deployment of durable resistance (R) genes. • Map-based cloning, transient expression in planta, pathogen transformation and DNA sequence variation across diverse isolates were used to identify and characterize PiAVR2 from potato late blight pathogen Phytophthora infestans. • PiAVR2 is an RXLR-EER effector that is up-regulated during infection, accumulates at the site of haustoria formation, and is recognized inside host cells by potato protein R2. Expression of PiAVR2 in a virulent P. infestans isolate conveys a gain-of-avirulence phenotype, indicating that this is a dominant gene triggering R2-dependent disease resistance. PiAVR2 presence/absence polymorphisms and differential transcription explain virulence on R2 plants. Isolates infecting R2 plants express PiAVR2-like, which evades recognition by R2. PiAVR2 and PiAVR2-like differ in 13 amino acids, eight of which are in the C-terminal effector domain; one or more of these determines recognition by R2. Nevertheless, few polymorphisms were observed within each gene in pathogen isolates, suggesting limited selection pressure for change within PiAVR2 and PiAVR2-like. • Our results direct a search for R genes recognizing PiAVR2-like, which, deployed with R2, may exert strong selection pressure against the P. infestans population.

Genome evolution following host jumps in the Irish potato famine pathogen lineage.

Many plant pathogens, including those in the lineage of the Irish potato famine organism Phytophthora infestans, evolve by host jumps followed by specialization. However, how host jumps affect genome evolution remains largely unknown. To determine the patterns of sequence variation in the P. infestans lineage, we resequenced six genomes of four sister species. This revealed uneven evolutionary rates across genomes with genes in repeat-rich regions showing higher rates of structural polymorphisms and positive selection. These loci are enriched in genes induced in planta, implicating host adaptation in genome evolution. Unexpectedly, genes involved in epigenetic processes formed another class of rapidly evolving residents of the gene-sparse regions. These results demonstrate that dynamic repeat-rich genome compartments underpin accelerated gene evolution following host jumps in this pathogen lineage.

In planta expression screens of Phytophthora infestans RXLR effectors reveal diverse phenotypes, including activation of the Solanum bulbocastanum disease resistance protein Rpi-blb2.

The Irish potato famine pathogen Phytophthora infestans is predicted to secrete hundreds of effector proteins. To address the challenge of assigning biological functions to computationally predicted effector genes, we combined allele mining with high-throughput in planta expression. We developed a library of 62 infection-ready P. infestans RXLR effector clones, obtained using primer pairs corresponding to 32 genes and assigned activities to several of these genes. This approach revealed that 16 of the 62 examined effectors cause phenotypes when expressed inside plant cells. Besides the well-studied AVR3a effector, two additional effectors, PexRD8 and PexRD36(45-1), suppressed the hypersensitive cell death triggered by the elicitin INF1, another secreted protein of P. infestans. One effector, PexRD2, promoted cell death in Nicotiana benthamiana and other solanaceous plants. Finally, two families of effectors induced hypersensitive cell death specifically in the presence of the Solanum bulbocastanum late blight resistance genes Rpi-blb1 and Rpi-blb2, thereby exhibiting the activities expected for Avrblb1 and Avrblb2. The AVRblb2 family was then studied in more detail and found to be highly variable and under diversifying selection in P. infestans. Structure-function experiments indicated that a 34-amino acid region in the C-terminal half of AVRblb2 is sufficient for triggering Rpi-blb2 hypersensitivity and that a single positively selected AVRblb2 residue is critical for recognition by Rpi-blb2.

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.