Genomes and virulence difference between two physiological races of Phytophthora nicotianae
- Hui Liu†1, 2,
- Xiao Ma†3,
- Haiqin Yu†4,
- Dunhuang Fang4,
- Yongping Li4,
- Xiao Wang1, 2,
- Wen Wang1,
- Yang Dong5, 3Email author and
- Bingguang Xiao4Email author
© Liu et al. 2016
Received: 15 September 2015
Accepted: 6 January 2016
Published: 28 January 2016
Black shank is a severe plant disease caused by the soil-borne pathogen Phytophthora nicotianae. Two physiological races of P. nicotianae, races 0 and 1, are predominantly observed in cultivated tobacco fields around the world. Race 0 has been reported to be more aggressive, having a shorter incubation period, and causing worse root rot symptoms, while race 1 causes more severe necrosis. The molecular mechanisms underlying the difference in virulence between race 0 and 1 remain elusive.
We assembled and annotated the genomes of P. nicotianae races 0 and 1, which were obtained by a combination of PacBio single-molecular real-time sequencing and second-generation sequencing (both HiSeq and MiSeq platforms). Gene family analysis revealed a highly expanded ATP-binding cassette transporter gene family in P. nicotianae. Specifically, more RxLR effector genes were found in the genome of race 0 than in that of race 1. In addition, RxLR effector genes were found to be mainly distributed in gene-sparse, repeat-rich regions of the P. nicotianae genome.
These results provide not only high quality reference genomes of P. nicotianae, but also insights into the infection mechanisms of P. nicotianae and its co-evolution with the host plant. They also reveal insights into the difference in virulence between the two physiological races.
KeywordsBlack shank Phytophthora nicotianae Genomes Hybrid assembly RxLR effector
Phytophthora nicotianae, also known as Phytophthora parasitica var. nicotianae, is a soil-borne bi-flagellated oomycete plant pathogen, which causes black shank in cultivated tobacco (Nicotiana tabacum), and root rot, leaf necrosis, and stem lesions in a variety of plants . P. nicotianae is able to infect a wide range of hosts, spanning 255 genera in 90 different plant families. It devastates the production of a number of economically important plants, and causes millions of dollars worth of economic losses each year in the tobacco industry alone [2, 3]. So far, management strategies for P. nicotianae are limited to non-host crop rotation, cultivation of pathogen-resistant breeds, and the use of chemical control (e.g. mefenoxam) . The primary reason for the difficulty in controlling P. nicotianae is the production and survival of chlamydospores in unfavorable growth conditions, as well as the production of motile zoospores. The ability of P. nicotianae to infect specific tobacco cultivars with different resistance genes defines four physiological races (0, 1, 2 and 3). The predominant physiological races, 0 and 1, are widely distributed throughout China, the United States and other major tobacco cultivation areas [5, 6]. Previous studies using tobacco cultivars with moderate or high levels of resistance have found that race 0 has better pathogenic and ecologic fitness levels than race 1, suggesting that the difference in virulence between the two races is affected by additional genetic factors . To discover better and more efficient ways to control the pathogen, we undertook a global examination of the genes involved in the infection process from different races. Although five strains of P. parasitica are already public available , none of these includes any of the four physiological races of P. nicotianae. Here we report the genomes of P. nicotianae physiological races 0 and 1, sequenced using a combination of PacBio single-molecule real-time (SMRT) sequencing technology, and Illumina HiSeq and MiSeq sequencing technologies, and identify candidate genes that may cause the difference in virulence between them.
Isolation of P. nicotianae races and genomic DNA extraction
Tobacco plants infected by either P. nicotianae race 0 or race 1 were obtained from Yunnan Tobacco Research Institute. Any surface dirt on the infected plant was washed off under tap water. After drying, stem tissue from the lesion margin were cut into 5 × 5 mm squares, sterilized using 70 % ethanol for 1 minute, and then rinsed three times using sterile water. Sterilized tissue squares were then placed in lima bean agar (LBA) plates amended with 50 μg/ml ampicillin, 100 μg/ml rifampicin, and 50 μg/ml of pentachloronitrobenzene to suppress possible contaminant. LBA plates were incubated for 2–3 days in darkness at 25 °C. Color and texture of the colony and mycelium were used to confirm the identity of P. nicotianae. Mycelium was transferred to LBA slants and cultured for 7 days in darkness at 25 °C. Genomic DNA was extracted using the modified cetyltrimethyl ammonium bromide method .
Sequencing and quality control
Sequencing and data size of P. nicotianae races 0 and race 1
Fragment size (bp)
Read length (bp)
Before quality control
After quality control
P. nicotianae race 0
Illumina mate pair
P. nicotianae race 1
Illumina mate pair
Genome and gene statistics of P. nicotianae races 0 and race 1
Longest size (kb)
Percentage of the assembly
P. nicotianae race 0
P. nicotianae race 1
Known transposable elements (TEs) were identified with RepeatMasker (version 3.2.6)  using the Repbase TE library (v16.10)  and default parameters. Tandem repeats were predicted using TRF . gypsy and copia types of long terminal repeat (LTR) were the main contributors to the repeat, making up 12.5 % and 3.5 % of the genome for race 0, and 11.5 % and 3.6 of the genome for race 1. For gene structure prediction, gene sets from 9 species including Phytophthora infestans , Phytophthora sojae , Phytophthora ramorum , Hyaloperonospora arabidopsis , Pythium aphanidermatum , Pythium arrhenomanes , Pythium irregulare , Pythium vexans , Pythium iwayamai  and Pythium ultimum  were used for homology-based prediction. GENSCAN , AUGUSTUS  and GlimmerHMM  were used for de novo gene prediction. Evidence derived from homology-based and de novo predictions were then integrated in GLEAN to generate a consensus gene set. A total of 17,797 and 14,542 protein-coding genes were annotated in P. nicotianae race 0 and race 1, respectively. Over 97 % of these genes could be aligned against KEGG , Swiss-Prot and TrEMBL databases . Mean exon numbers per gene in P. nicotianae and related species varied between 2.2 and 2.8, suggesting that homology and de novo-based prediction were appropriate for annotation (Additional file 1). We also used publicly available expressed sequence tags (ESTs) from the appressorium  and mycelium [36, 37] of P. nicotianae to validate the annotation. We retrieved a total of 10,524 ESTs from the dbEST database. Using the threshold of match length >200 bp and E-value <1e-5, we aligned 8,043 ESTs to the race 0 genome and 7,618 ESTs to the race 1 genome. Additionally, 4,454 genes in race 0 and 3,604 genes in race 1 were supported by at least one EST (Additional file 2). Whole genome comparison using NUCmer  found that average identity was 99 % for 1-to-1 alignment, and 98.84 % for m-to-m alignment between P. nicotianae races 0 and 1. Using KaKs_Calculator, mean synonymous mutation ratio (Ks) was estimated to be 0.075 between race 0 and race 1 , and four genes were identified to be positively selected (Additional file 3).
Gene family clustering and evolution
ABC transporter expanded in P. nicotianae
The ATP-binding cassette transporter (ABC transporter) superfamily facilitates the transport of ions, proteins, lipids and toxins across plant membranes . Interestingly, a domain-centric study found this gene family to be enriched in the oomycete plant pathogen genomes . It was proposed that an important function of ABC transporters in pathogens involves exporting toxic phytoalexins [44, 45]. Based on the result of CAFÉ analysis, we found the ABC transporter gene family to be significantly expanded in the branch of P. nicotianae (likelihood ratio test, p-value < 0.05), but not in the branch of P. infestans (likelihood ratio test, p = 0.9). To verify this result, we used Pfam to annotate ABC transporter domains (PF00005.22, PF00664.18, PF01061.19) between P. infestans, and P. nicotianae races 0 and 1 (Additional file 6). The portions of ABC transporters in P. nicotianae were significantly larger than that in P. infestans (chi-square test, p < 0.05). This result suggests that the ABC transporter family plays important roles in P. nicotianae in its adaptive evolution to the host.
Distribution of effectors and their differences in races 0 and 1
Plant pathogens have evolved to secrete effectors, which can manipulate the host immune system and suppress host defense. Based on their target sites in the host plant, effectors can be classified into two classes: (1) apoplastic effectors, which are secreted into plant extracellular spaces; and (2) cytoplasmic effectors, which are translocated into the plant cell. Some effector genes, e.g. ATR5 in H. arabidopsidis, are found to be avirulence genes . These genes are under selective pressure to evade host recognition while maintaining their original functions.
Crinkler (CRN) effectors are another important class of effectors that cause leaf crinkling in plants . To investigate CRN effectors in P. nicotianae races 0 and 1, we first used EMBOSS getorf (−minsize 300) to extract open reading frames (ORFs) from the whole genome, and then used HMMer (−E 1e-5) with existing profiles . Predicted CRN effectors were filtered by the presence of the LxLFLAK motif. A total of 32 and 26 CRN effectors were annotated in P. nicotianae races 0 and 1, respectively. However, the number of CRN effectors may be underestimated, given the model we used .
Availability of supporting data
The genome assembly, annotation and sequencing reads of each sequencing library are available in the NCBI repository, project ID PRJNA294216. The genome assembly and annotation can also be accessed via the GigaScience GigaDB database .
- ABC transporter:
ATP-binding cassette transporter
expressed sequence tag
lima bean agar
long terminal repeat
open reading frame
effectors with Arg-X-Leu-Arg motif
single-molecular real-time sequencing
We owe sincere thanks to Dr. Chen Wei for his pre-submission review of the article. We thank Yunnan Tobacco Research Institute for kindly providing infected tobacco plants. We thank Hou Yujie for help on gene annotation and Dr. Shi Xiaofei for useful discussions of the article.
This work was supported by grants from the China National Tobacco Corporation (110201201003 [JY-03]) and 110201301006 [JY-06]), and Yunnan Tobacco Corporation (2012YN01 and 2013YN01).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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