Guohong Cai

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

The family narnaviridae: simplest of RNA viruses.

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 ∼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 ∼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.

A member of the virus family Narnaviridae from the plant pathogenic oomycete Phytophthora infestans.

Members of the virus family Narnaviridae contain the simplest genomes of any RNA virus, ranging from 2.3 to 3.6 kb and encoding only a single polypeptide that has an RNA-dependent RNA polymerase domain. The family is subdivided into two genera based on subcellular location: members of the genus Narnavirus have been found in the yeast Saccharomyces cerevisiae and in the oomycete Phytophthora infestans and are confined to the cytosol, while members of the genus Mitovirus have been found only in filamentous fungi and are found in mitochondria. None identified thus far encodes a capsid protein; like several other RNA viruses of lower eukaryotes, their genomes are confined within lipid vesicles. As more family members are discovered, their importance as genetic elements is becoming evident. The unique association of the genus Mitovirus with mitochondria renders them potentially valuable tools to study biology of lower eukaryotes.

Genome-wide microsatellite identification in the fungus Anisogramma anomala using Illumina sequencing and genome assembly.

A virus that has properties consistent with inclusion in the virus family Narnaviridae was described in Phytophthora infestans, the oomycete that caused the Irish potato famine. The genome of phytophthora infestans RNA virus 4 (PiRV-4) is 2,984 nt with short complementary terminal sequences and a single open reading frame predicted to encode an RNA-dependent RNA polymerase (RdRp) most closely related to saccharomyces cerevisiae narnavirus 20S (ScNV-20S) and ScNV-23S, the members of the genus Narnavirus, family Narnaviridae. This report constitutes the first description of a member of the family Narnaviridae from a host taxon outside of the kingdom Fungi.

A novel virus of the late blight pathogen, Phytophthora infestans, with two RNA segments and a supergroup 1 RNA-dependent RNA polymerase.

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N50 of 10,384 bp and scaffold N50 of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery.

A new virus from the plant pathogenic oomycete Phytophthora infestans with an 8 kb dsRNA genome: The sixth member of a proposed new virus genus

Double-stranded RNA representing four distinct electrophoretic patterns was found in a screen of Phytophthora infestans isolates. Two dsRNAs that always appeared together were sequenced. RNA 1, which was 3160 nt plus a poly (A) tail, contained a single deduced ORF with the potential to encode a polyprotein of 977 aa with motifs characteristic of supergroup I viral RdRps. The 2776 nt, polyadenylated RNA2 contained an ORF with a potential to encode a polyprotein of 847 aa including a possible trypsin-like serine protease, and a second putative ORF of unknown function. An alternative form of RNA2, in which a 19-nt stretch was replaced by a 9-nt sequence, was detected in 4 of 17 clones sequenced. Based on genome structure and phylogenetic analysis, this virus did not fit into any known virus family and we tentatively named it Phytophthora infestans RNA virus 1 (PiRV-1).

Gene profiling in partially resistant and susceptible near‐isogenic tomatoes in response to late blight in the field

A virus designated Phytophthora infestans RNA virus 3 (PiRV-3) was characterized from an isolate of P. infestans that was co-infected with a second previously described virus, PiRV-4, a member of the virus family Narnaviridae (Cai et al., 2012). The genome of PiRV-3 is 8112 nt and one strand, designated the positive strand, has two deduced overlapping open reading frames linked by a potential frameshift sequence. The first open reading frame (ORF1) is predicted to encode a protein of unknown function, and ORF2 is predicted to encode an RNA-dependent RNA polymerase (RdRp) most closely related to six unclassified dsRNA viruses of filamentous fungi. The genome organizations of five of the related viruses are similar to PiRV-3, indicating taxonomic linkage among those viruses. We suggest that PiRV-3 and related viruses should be collected into a new virus taxon.

Chapter Twelve - Phytophthora Viruses

In order to better understand resistance to Phytophthora infestans in tomato, we compared the global gene expression of the susceptible tomato, M82, with its more resistant near-isogenic line, 6-2 (IL6-2), under field conditions using a microarray with more than 12 800 tomato expressed sequence tags (ESTs). Because variance in the field was a major concern, we investigated the likelihood of false positives or false negatives and demonstrated that either probability was very low. The two isolines had indistinguishable constitutive gene expressions prior to inoculation. However, a few genes were particularly prone to variation in both isolines in the absence of P. infestans. Included among these genes were catalase, genes coding for pathogenesis-related proteins, endochitinase and cytochrome P450. In response to inoculation with P. infestans, a time course of gene expression identified 1248 transcripts that were similarly induced or repressed in each line, and 991 that were differentially expressed between the two lines. These differences provide hypotheses to explain the difference in resistance between the two isolines. For example, one hypothesis is that genes up-regulated in IL6-2 in response to inoculation with P. infestans, but not up-regulated in M82, contribute to the resistance in IL6-2. Using virus-induced gene silencing (VIGS), we were able to partially silence two such genes-one encoded a protein with homology to an R gene with the Toll/interleukin-1 receptor-nucleotide-binding site-leucine-rich repeat (TIR-NBS-LRR) motif (37O19) and the other encoded a peroxisomal membrane protein (35P7). Partial silencing of 37O19 reduced the resistance in IL6-2 (P = 0.001), but had no effect on the response of M82. Partial silencing of 35P7 reduced the resistance in IL6-2 moderately significantly (P = 0.067), but had no effect in M82. We expect that hypotheses developed from this gene expression study performed under field conditions will provide an important avenue to an accurate understanding of the genes involved in resistance.

Phytophthora infestans RNA virus 2, a novel RNA virus from Phytophthora infestans, does not belong to any known virus group

Phytophthora sp. is a genus in the oomycetes, which are similar to filamentous fungi in morphology and habitat, but phylogenetically more closely related to brown algae and diatoms and fall in the kingdom Stramenopila. In the past few years, several viruses have been characterized in Phytophthora species, including four viruses from Phytophthora infestans, the late blight pathogen, and an endornavirus from an unnamed Phytophthora species from Douglas fir. Studies on Phytophthora viruses have revealed several interesting systems. Phytophthora infestans RNA virus 1 (PiRV-1) and PiRV-2 are likely the first members of two new virus families; studies on PiRV-3 support the establishment of a new virus genus that is not affiliated with established virus families; PiRV-4 is a member of Narnaviridae, most likely in the genus Narnavirus; and Phytophthora endornavirus 1 (PEV1) was the first nonplant endornavirus at the time of reporting. Viral capsids have not been found in any of the above-mentioned viruses. PiRV-1 demonstrated a unique genome organization that requires further examination, and PiRV-2 may have played a role in late blight resurgence in 1980s-1990s.