Transgenerational gene silencing causes gain of virulence in a plant pathogen.

Avirulence (Avr) genes of plant pathogens encode effector proteins that trigger immunity in plants carrying appropriate resistance (R) genes. The Phytophthora sojae Avr3a gene displays allelic variation in messenger RNA transcript levels. P. sojae strains with detectable Avr3a gene transcripts are avirulent on plants carrying the R-gene Rps3a, whereas strains lacking Avr3a mRNA escape detection by Rps3a and are virulent. Here we show non-Mendelian interactions between naturally occurring Avr3a alleles that result in transgenerational gene silencing, and we identify small RNA molecules of 25 nucleotides that are abundant in gene-silenced strains but not in strains with Avr3a mRNA. This example of transgenerational gene silencing is exceptional because it is naturally occurring and results in gain of virulence in a pathogenic organism.

Plant pathogens secrete effector proteins to enable disease, but evolution favors host plant immune systems to recognize these factors as cues for activating rapid defence responses that arrest the infection1. Effector-triggered immunity in plants is the consequence of signalling events that are determined by resistance (R) gene products interacting, directly or indirectly, with pathogen avirulence (Avr) effectors.

Phytophthora sojae causes stem and root rot of soybean and is a major disease problem that plagues this crop2. The P. sojae Avr3a gene encodes a predicted protein of 111 amino acids that includes a signal peptide, a host-targeting motif and a carboxy-terminal effector domain, which are common features of oomycete Avr effectors3456. Avr3a resides in a 10.8 kilobase (kb) DNA segment that displays copy number variation among P. sojae strains56. Four haplotypes of Avr3a are known and gain of virulence on the R-gene Rps3a is caused by messenger RNA transcript differences between strains. In crosses between P. sojae strains P6497 (Avr3a/Avr3a) and P7064 (Avr3a/Avr3a), avirulence towards Rps3a plants segregates as a dominant Mendelian trait conditioned by the presence of Avr3a. Transcripts of Avr3a but not Avr3a mRNA are detectable in each of the parental strains and their progeny56.

Sequencing of Avr3a alleles and analysis of mRNA transcripts in 17 different P. sojae strains revealed four different haplotypes6, but the inheritance behaviour of each of these haplotypes in segregating populations has not been investigated. The present study was intended to test the inheritance Avr3a in P. sojae strains with haplotypes that are different from strains P6497 and P7064. To our surprise, we discovered that genetic outcrosses between P. sojae strains ACR10 and P7076 resulted in transgenerational gene silencing of Avr3a and gain of virulence on soybean plants carrying the Rps3a gene.

Results

Transgenerational gene silencing of Avr3a

To determine the inheritance of other Avr3a alleles, we created crosses between P. sojae strains P7076 (Avr3a/Avr3a) and ACR10 (Avr3a/Avr3a). The strain P7076 is avirulent, whereas strain ACR10 is virulent on Rps3a plants, and mRNA transcripts are detectable for Avr3a but not Avr3a, as we have previously shown56 (Fig. 1a). To select hybrid progeny and follow segregation of Avr3a alleles we used co-dominant DNA markers derived from cleaved amplified polymorphic (CAP) fragments, because Avr3a and Avr3a differ in sequence56. A total of 28 F1 hybrid (Avr3a/Avr3a) progeny were isolated, scored for virulence on Rps3a plants and assessed for the presence of Avr3a mRNA transcripts by reverse transcriptase polymerase chain reaction (RT–PCR) (Table 1, Fig. 2, Supplementary Fig. S1). All 28 F1 progeny were virulent on Rps3a plants and were lacking Avr3a mRNA transcripts. This result was unexpected because heretofore avirulent alleles of Avr3a that accumulate mRNA transcripts were dominant to virulent alleles that lack transcripts, such as in crosses between P. sojae strains P6497 and P7064. To further explore the segregation of Avr3a alleles, virulence and mRNA transcript accumulation in this cross, we generated three different F2 populations from separate F1 individuals. In all, 139 F2 progeny were tested for segregation of Avr3a alleles and mRNA transcripts and scored for virulence on Rps3a plants. The Avr3a and Avr3a alleles segregated normally in each F2 population but remarkably all 139 F2 progeny lacked Avr3a mRNA transcripts and were virulent on Rps3a plants (Table 1). A sampling of eight F3 individuals, derived from homozygous Avr3a/Avr3a F2 cultures, indicates that gene silencing is maintained and meiotically stable in these inbred lines (Supplementary Fig. S2). These results demonstrate heritable transgenerational gene silencing of Avr3a in crosses between P. sojae strains ACR10 and P7076.

Figure 1

Characteristics of P. sojae Avr3a alleles and deep sequencing of sRNA.

(a) A schematic illustration of Avr3a alleles from four different strains of P. sojae. The segment of DNA harbouring Avr3a and four other predicted genes is shown. Predicted open reading frames are indicated by bold arrows. Shown for each allele is the copy number of the DNA segment, presence (+) or absence (−) of Avr3a mRNA transcripts and disease outcome on Rps3a plants as virulent (V) or avirulent (A). (b) Profiles of sRNA from P. sojae strains ACR10 and P7076, and from a pool of F2 cultures selected for their Avr3a/Avr3a genotype. Average depth of coverage across the 10.8-kb genome segment containing Avr3a was normalized to the total number of trimmed reads for each sample. (c) Size of sRNA aligning to the 10.8-kb DNA segment. The total number of counts of RNA sequence reads and the size in nucleotides (nt) is shown for P. sojae strain ACR10.

Table 1

Segregation of Avr3a alleles and virulence phenotypes in F1 and F2 populations (ACR10 × P7076) of Phytophthora sojae.

Population	n	Avr3a allele genotype			P	Virulence		mRNA
		Avr3aACR10Avr3aACR10	Avr3aACR10Avr3aP7076	Avr3aP7076Avr3aP7076		A	V	(+)	(−)
F1	28	0	28	0	1.0	0	28	0	28
F2-1	51	16	26	9	0.38	0	51	0	51
F2-2	44	14	16	14	0.19	0	44	0	44
F2-3	44	10	22	12	0.91	0	44	0	44

The number of progeny (n) in each population and the P value from a χ2-test for Mendelian segregation of genotypes is shown. Virulent (V) and avirulent (A) phenotypes were scored by inoculation of plants, and the presence (+) or absence (−) of mRNA transcripts of Avr3a as determined by RT–PCR.

Figure 2

Photographs of P. sojae-infected soybean plants showing disease outcomes.

Virulence assays of P. sojae cultures were performed on greenhouse-grown seedlings of soybean cultivar Williams (rps3a) and the isoline L83-570 (Rps3a) by hypocotyl inoculation with P. sojae mycelia. Shown are disease outcomes 6 days after inoculation of plants with the parental strains, the F1 hybrids used to construct each of the three F2 populations, or with representative F2 individuals illustrating each Avr3a genotype from the three F2 populations tested. Pots are 10 cm in diameter.

Distinctive profile of small RNA (sRNA) in silenced strains

To explore the cause of transgenerational silencing of Avr3a, we performed deep sequencing of sRNA from each parental P. sojae strain ACR10 and P7076, and from a sample of F2 cultures with the genotype Avr3a/Avr3a. Results from this analysis show that the profile of sRNA coverage across the Avr3a region differs between ACR10 and P7076 (Fig. 1b). The sRNA sequences matching to the Avr3a region are far more abundant in strain ACR10 compared with P7076 (Table 2). The most prevalent of these sRNA molecules are 24–26 nucleotides in length (Fig. 1c). In strain ACR10, there is a nearly continuous coverage of sRNA sequences that align to a 3.7 kb span of the Avr3a interval including the 5′ and 3′ intergenic segments but not extending into flanking genes. In contrast, in strain P7076 coverage of matching sRNA is limited to a 0.3-kb segment occurring in the 5′ intergenic region. Sequencing of sRNA from homozygous Avr3a/Avr3a F2 cultures resulted in a profile of coverage that is nearly identical to that from strain ACR10 (Avr3a/Avr3a) rather than that from P7076 (Avr3a/Avr3a). The sRNA profiles were replicated from independent samples of ACR10, P7076, F1 and F2 cultures, and extended to include samples from additional P. sojae strains P6497 and ACR16 (Table 2; Supplementary Fig. S3). The results consistently show a pattern of extensive sRNA coverage of Avr3a occurs in gene-silenced strains.

Table 2

Deep sequencing of sRNA from Phytophthora sojae. *

Sample†	Avr3a genotype	Avr3a mRNA‡	Virulence on Rps3a	Total number of trimmed sRNA sequence reads	Matches to Avr3asegment§	Normalized matches per 106 reads
Parental strains
P7076 (rep1)	Avr3aP7076/Avr3aP7076	+	A	4,640,922	306	65
P7076 (rep2)	Avr3aP7076/Avr3aP7076	+	A	13,040,733	659	50
ACR10 (rep1)	Avr3aACR10/Avr3aACR10	−	V	3,338,276	1,715	513
ACR10 (rep2)	Avr3aACR10/Avr3aACR10	−	V	13,164,585	9,488	720
Progeny
F1-2	Avr3aACR10/Avr3aP7076	−	V	48,505,836	10,603	218
F2-2-14	Avr3aACR10/Avr3aACR10	−	V	50,286,928	30,809	612
F2-2-26	Avr3aACR10/Avr3aP7076	−	V	45,921,916	17,812	387
F2-2-35	Avr3aP7076/Avr3aP7076	−	V	45,141,247	12,883	285
F2 pool (rep1)	Avr3aP7076/Avr3aP7076	−	V	14,712,321	4,411	299
F2 pool (rep2)	Avr3aP7076/Avr3aP7076	−	V	20,495,465	6,204	302
Other strains
P6497	Avr3aP6497/Avr3aP6497	+	A	17,919,740	345	19
ACR16	Avr3aACR16/Avr3aACR16	−	V	20,450,286	3,338	163

*Samples of sRNA were purified from mycelia cultures of P. sojae for library construction and sequencing, as described in Methods section.

†Source of all P. sojae strains and sequence of Avr3a alleles has previously been described56.

‡Presence (+) or absence (−) of Avr3a mRNA, as determined by RT–PCR.

§Matches of sRNA sequences to 10.8 kb Avr3a segment, as shown in Fig. 1.

A, avirulent; rep, replicate; V, virulent.

Discussion

Previous studies have shown that transformation of organisms, including species of Phytophthora, with recombinant DNA or RNA molecules can cause internuclear or trangenerational gene silencing that resembles our observations of naturally occurring silencing of Avr3a (refs 7,8). Moreover, results from comparative genome sequencing of species within the Phytophthora infestans clade suggest a role for epigenetic mechanisms in the rapid evolution of Phytophthora species9. Thus, we propose that transgenerational gene silencing of Avr3a in P. sojae is the result of epigenetic changes that modulate the expression state of this gene, and that are transmitted to progeny in a non-Mendelian fashion.

Given the emerging functions of sRNAs in controlling epigenetic marks and gene expression states in numerous experimental systems, it is noteworthy that we found an association between sRNAs and gene silencing at the Avr3a locus. In plants, sRNA of 24–26 nucleotides are involved in directing chromatin changes including DNA methylation, whereas shorter ones of 21–22 nucleotides participate in mRNA degradation10. Heterochromatic silencing in yeast is initiated by an interaction of sRNA and RNA polymerase II, which guides histone modifications that control chromatin structure and gene expression11. Thus, the evidence suggests a role for sRNA in directing the transgenerational gene expression changes of the P. sojae Avr3a gene.

Our observations of transgenerational gene silencing of Avr3a provide a remarkable example of pathogen adaptation to host immune surveillance. The interactions between pathogens and their hosts are often interpreted as an evolutionary race, where pathogen virulence and host resistance are at the forefront12. Transgenerational gene silencing of an Avr effector locus represents a novel method of host immune evasion that offers the ability to spread quickly in pathogen populations, but yet is potentially reversible as the sequence of the Avr gene itself remains unchanged. We predict that other eukaryotic pathogens of plants, humans and animals will employ similar mechanisms to escape detection by host immune systems.

Methods

Phytophthora sojae strains and crosses

The origin of P. sojae strains and methods of culture, propagation and oospore isolation have been described56131415. P. sojae is an oomycete and diploid microorganism that reproduces sexually through the development of oogonia (female gametophyte) and antheridia (male gametophyte). P. sojae is homothallic and is not known to have mating types, therefore oospores can develop from self-fertilization or from out-crossing when different strains of the organism are grown together2. To perform sexual crosses of P. sojae, parental strains are co-cultivated and oospores are isolated from culture homogenates by a series of purification steps131415. Purified oospores are diluted and plated out on water agar to germinate. Individual germinating oospores are picked under a dissecting microscope and transferred to rich growth medium. Samples of purified DNA from the resulting cultures are tested using co-dominant DNA markers polymorphic between the parents, to determine whether the individuals result from self-fertilization or from out-crossing events between the parental strains. Such markers designed to distinguish Avr3a and Avr3a alleles (described below) were used to identify F1 progeny in crosses between strain ACR10 and P7076. The F2 populations were generated by isolating oospores from self-fertilized F1 individuals.

Purification of nucleic acids and scoring of markers and transcripts for Avr3a

Mycelia cultures of P. sojae for nucleic acid isolation were grown on vegetable juice (V8) agar media overlaid with cellophane (BioRad). After growth in darkness for 7 days at 25 °C, mycelia-grown cellophane disks were peeled off the media, frozen at −80 °C and ground to a fine powder in liquid N2 using a mortar and pestle. Genomic DNA was purified using conventional phenol–chloroform extraction procedures followed by precipitation with isopropanol16. Purfied DNA pellets were washed with 70% (v/v) ethanol, dried and resuspended in a solution of 10 mM Tris HCl, pH 7.4, 0.1 mM EDTA. CAP markers were used to track segregation of the Avr3a and Avr3a alleles. For CAPs analysis, PCR amplification was performed with 5 ng of genomic DNA as template in a volume of 25 μl under the following conditions: 0.2 mM dNTPs, 1.5 mM MgCl2, 0.4 μM of each primer (forward primer 5′-GCTGCTTCCTTCCTGGTTGC-3′ and reverse primer 5′-GCTGCTGCCTTTTGCTTCTC-3′), 1.25 U Taq DNA polymerase and 1 × PCR buffer as recommended by the supplier (Invitrogen, Life Technologies). The amplification was carried out in a 96-well plate (GeneAmp PCR System 9700, Applied Biosystems) under the following conditions: 94 °C for 2 min; 40 cycles of 94 °C for 40 s, 58 °C for 40 s, 72 °C for 2 min; followed by an extension step of 10 min at 72 °C. For restriction analysis of the PCR fragments, 10 μl of the amplified products were incubated overnight at 37 °C in a volume of 20 μl using 3 U of AluI (New England Biolabs). Digestion products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining according to standard methods16. The Avr3a sequence includes an AluI site, whereas the Avr3a sequence lacks the restriction site and is not digested56.

Total RNA was extracted from frozen, pulverized mycelia samples in a solution of phenol-guanidine isothiocyanate (TRIzol, Invitrogen) according to instructions provided by the manufacturer. Approximately 1 μg total RNA was subject to DNaseI (Invitrogen) treatment before RT–PCR, as previously described56. Cycling conditions for detecting transcripts of Avr3a as well as for the P. sojae actin gene (forward primer 5′-CGAAATTGTGCGCGACATCAAG-3′ and reverse primer 5′-GGTACCGCCCGACAGCACGAT-3′), used as a control, were described as above with the exception that 25 cycles were used for transcript analysis.

Plant inoculation and virulence assays

Virulence assays of P. sojae cultures were performed on greenhouse-grown seedlings of soybean cultivar Williams (rps) and the isoline L83-570 (Rps3a), according to standard methods56. Soybean seeds (20–30 seeds per pot) were sown in 10 cm pots and grown for 7 days before disease testing. For inoculation, P. sojae cultures were grown for 5–7 days on 0.9% (v/v) V8 agar plates. Inoculum was prepared by excising 1 cm segments of agar cut from the growing edge of mycelia colonies and macerating the cultures through a 3-ml syringe attached to an 18-gauge needle. Soybean plants were inoculated in the mid-section of each hypocotyl by making a small incision for injection of the mycelial slurry. Inoculated plants were covered with plastic bags to maintain humidity for 2 days. Disease symptoms were allowed to develop for an additional 4 days before phenotypes were scored. A minimum of three independent replicates of the disease assay were performed for each P. sojae culture tested.

Deep sequencing of P. sojae sRNA

To profile sRNA molecules of P. sojae cultures, samples of RNA were purified from mycelia cultures as described above. The size selection of sRNA, attachment of adaptor molecules and library construction was performed using prepared supplies and reagents (Illumina small RNA v1.5), following instructions provided by the manufacturer. Sequencing was performed on a flow cell instrument (Illumina GAII). After trimming to remove adaptor sequences and low-quality sequences, the depth of sRNA coverage of the Avr3a region was determined by alignment, and normalized to the number of reads per library. Average depth of coverage across the 10.8-kb genome segment containing Avr3a was determined using a 50-base pair moving interval and values normalized to the total number of trimmed reads for each sample. The number of trimmed reads obtained for each of the sequenced sRNA libraries is proved in Table 2.

Author contributions

D.Q. and M.G. conceived and designed the experiments. D.Q. performed the experiments. D.Q., B.P.C. and M.G. analysed the data. M.G. wrote the paper.

Additional information

How to cite this article: Qutob, D. et al. Transgenerational gene silencing causes gain of virulence in a plant pathogen. Nat. Commun. 4:1349 doi: 10.1038/ncomms2354 (2013).