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 19th 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 epidemic

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

<i>P. infestans</i> population displacement in Great Britain by the <i>13_A2</i> genotype.

<p>(A) Frequency of multilocus genotypes (MLGs) over the course of 11 years from more than 4000 potato blight outbreaks. The number of isolates fingerprinted each year and dominant MLGs of each mating type are indicated. Isolates of MLGs that occurred at a very low frequency in a single year are grouped under the category termed <i>‘misc’</i>. The shading between the bars indicates the proportion of A1 and A2 mating type isolates. (B) Minimum Spanning Trees based on the alleles at 11 SSR loci indicating the relatedness of the main MLGs and decrease in population diversity between the periods 2003–5 and 2008. The numerous short branches from the <i>13_A2</i> MLG node reflect the high mutation rate in some SSR markers that results in intra-MLG diversity (<i>n</i> is the number of isolates from which the trees are derived). (C) Spatial pattern of spread two dominant MLGs across Great Britain (GB) from 2006–2008 (the numbers of isolates are indicated on each pie chart).</p

Summary of nonsynonymous and synonymous single nucleotide polymorphisms (SNPs) in coding genes (CDSs) of <i>P. infestans</i> 06_3928A compared to T30-4 reference genome strain.

*<p>count of SNPs causing loss of stop codons were omitted;</p>†<p>dN/dS rates were calculated using Yang method reported in Yang and Nielsen <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002940#ppat.1002940-Yang2" target="_blank">[56]</a>.</p><p>Nonsynonymous and synonymous SNPs were calculated for all genes, core orthologs and RXLRs. Core orthologs as genes showing orthologous sequences 1∶1∶1 in <i>P. infestans: P. ramorum: P. sojae</i> genomes respectively <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002940#ppat.1002940-Haas1" target="_blank">[23]</a>.</p

Gene expression polymorphisms correlate with extended biotrophy in <i>P. infestans 13_A2</i> isolate 06_3928A.

<p>(A) Number of genes (left) and RXLR effector genes (right) that are induced during potato infection in <i>P. infestans</i> T30-4, 06_3928A and NL07434 strains. Only a small subset of genes is consistently induced in the three strains. (B) Average gene expression pattern during potato infection for all genes (left) and RXLR effector genes (right) induced in all three <i>P. infestans</i> strains analyzed. A consistent divergence is observed at 3 days post inoculation (dpi) when gene induction is maintained in the 06_3928A isolate only. (C) Number of all (left) and RXLR effector genes (right) induced at various time points during potato infection in each of the three <i>P. infestans</i> strains. Compared to other strains, 06_3928A shows the highest number of genes that are induced both at 2 and 3 dpi. (D) Variation in the size of the biotrophic area (infected living host tissue) in lesions induced by three <i>P. infestans</i> strains during potato infection. Error bars are s.e.m. over 28 measurements at 2, 3 and 4 dpi. Representative pictures illustrate the sizes of the necrotrophic (infected dead host tissue, centre of the lesion) and biotrophic (periphery of the lesion, lighter grey ring) growth (mm) with the respective <i>P. infestans</i> strain in color (blue for 06_3928A, red for T30-4 and orange for NL07434 strain) from lesions at 3 dpi.</p

<i>13_A2</i> genotype is among the most aggressive <i>P. infestans</i> genotypes on potato.

<p>Aggressiveness of 26 <i>P. infestans</i> isolates grouped into 10 multilocus genotypes (MLGs) on leaves of five potato cultivars (A to E) estimated using mean latent period (x-axis) and mean lesion size at 6 days post inoculation (dpi) (y-axis). Measurements made at 13°C and 18°C are indicated with empty and filled symbols, respectively, and the three most aggressive MLGs colour-coded. (F) The sum of the ranked positions of each MLG according to lesion size at 13°C and 18°C indicates that <i>6_A1</i> and <i>13_A2</i> isolates more often had the largest lesions (particularly at 13°C). The standard errors (s.e.) and degrees of freedom (d.f.) for cultivar by MLG comparisons of latent period and lesion size at 13°C and 18°C (charts A to E) are shown in the lower corner of the figure.</p

Distribution of polymorphism in genes of <i>P. infestans 13_A2</i> isolate 06_3928A.

<p>Y-axis indicates the frequency of synonymous substitutions (dS) in (A) and nonsynonymous substitutions (dN) in (C) given for RXLR genes, other (non-RXLR) genes and core ortholog genes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002940#ppat.1002940-Haas1" target="_blank">[23]</a>. Y-axis shows the rates of synonymous substitutions (dS) in (B) and nonsynonymous substitutions (dN) in (D) given for RXLR genes, other (non-RXLR) genes and core ortholog genes. Box and whisker plots in (B and D) show median, first and third quartile, and first values beyond 1.5 times the interquatile range.</p

Field aggressiveness of five <i>P. infestans</i> genotypes estimated using mark-and-recapture methods during a controlled blight epidemic.

<p>The proportion of each multilocus genotypes (MLG) recovered amongst 716 foliar blight lesions over a 21 day epidemic initiated with 5 <i>P. infestans</i> isolates of known MLG presented by (A), sampling date and (B), Cultivar. Note; in each case the Y-axis scale is set from 0.7 to 1.0 to more clearly reveal the proportion of the non-<i>13_A2</i> MLGs. The isolates of ‘Other’ MLGs were of non-introduced clonal MLGs that migrated into the trial.</p