two rxlr avirulence genes in phytophthora sojae determine soybean ...

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Title: 2

Two RxLR avirulence genes in Phytophthora sojae determine soybean Rps1k-3

mediated disease resistance 4

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Tianqiao Song1*, Shiv D. Kale2*, Felipe D. Arredondo2,3*, Danyu Shen1,3, Liming Su1, 6

Li Liu1, Yuren Wu1, Yuanchao Wang1, Daolong Dou1,2 and Brett M. Tyler2, 3† 7

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1 College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China 10

2 Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061, USA 11

3 Current address: Center for Genome Research and Biocomputing and Department of 12

Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA 13

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* These authors made equal contributions 15

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† To whom correspondence should be addressed. Email: [email protected]; 17

phone: 541-737-3347; FAX: 541-737-3045. 18

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The author responsible for distribution of materials integral to the findings presented in 20

this article is: Brett Tyler ([email protected]). 21

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Submission data: Submitted to GenBank, accession numbers KC312950-KC312957. 23

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Summary 1

Rps (resistance to Phytophthora sojae) genes have been widely used in soybean against 2

root and stem rot diseases caused by this oomycete. Among 15 known soybean Rps 3

genes, Rps1k has been the most widely used in the past four decades. Here, we show that 4

the products of two distinct but closely linked RxLR effector genes are detected by 5

Rps1k-containing plants, resulting in disease resistance. One of the genes is Avr1b-1, that 6

confers avirulence in the presence of Rps1b. Three lines of evidence, including over-7

expression and gene silencing of Avr1b-1 in stable P. sojae transformants, as well as 8

transient expression of this gene in soybean, indicated that Avr1b could trigger an Rps1k-9

mediated defense response. Some isolates of P. sojae that do not express Avr1b are 10

nevertheless unable to infect Rps1k plants. In those isolates we identified a second RxLR 11

effector gene (Avr1k, designated Avr1k), located 5 kb away from Avr1b-1. Silencing or 12

over-expression of Avr1k in P. sojae stable transformants resulted in the loss or gain, 13

respectively, of the avirulence phenotype in the presence Rps1k. Only isolates of P. sojae 14

with mutant alleles of both Avr1b-1 and Avr1k could evade perception by the soybean 15

plants carrying Rps1k. 16

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Introduction 1

The oomycete genus Phytophthora contains over 120 species, most of which are 2

destructive plant pathogens (Erwin and Ribiero, 1996; Kroon et al. 2012). Oomycetes 3

form a distinct phylogenetic lineage of fungus-like eukaryotic microorganisms (Kamoun, 4

2003; Tyler, 2006), related to diatoms. Phytophthora sojae (Kaufmann and Gerdeman 5

1958) was identified as a fast spreading and devastating root rot pathogen of soybean 6

(Glycine max L. Merr.) in the United States and Canada in the early 1950s (Kaufmann 7

and Gerdemann 1958). Since then, the disease has become endemic in almost all soybean 8

production regions around the globe. In the US losses due to P. sojae have risen steadily 9

and in 2008 – 2010 averaged around $400 million (Wrather and Koenning, 2010). In 10

China, the pathogen is a new and invasive problem in large scale soybean growing 11

regions, such as Heilongjiang province (Cui et al. 2010). 12

Resistance conferred by single dominant Rps (resistance to P. sojae) genes has 13

been providing generally effective protection of soybean against this pathogen (Tyler 14

2008). To date, 15 Rps genes have been mapped to nine genetic locations (Polzin et al. 15

1994; Lohnes and Schmitthenner 1997; Bhattacharyya et al. 2005; Sandhu et al. 2005; 16

Gordon et al. 2006; Wu et al. 2011), including five alleles of Rps1 (Rps1a. Rps1b, Rps1c, 17

Rps1d and Rps1k) and three alleles of Rps3 (Rps3a, Rps3b and Rps3c). Rps1k confers 18

stable resistance against most races of P. sojae and has been widely used for the past four 19

decades in North America, indicating that its corresponding avirulence gene(s) in the 20

pathogen were not easily lost, even under high selection pressure. 21

The Rps1 locus contains numerous genes encoding coiled coil-nucleotide binding-22

leucine rich repeat (CC-NB-LRR) proteins, and these genes show considerable variation 23

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in numbers and arrangement among different Rps1 alleles (Bhattacharyya et al. 2005; 1

Gao et al. 2008). Several of these genes have been cloned from the Rps1k allele of the 2

Rps1 locus and two of them, Rps1k-1 and Rps1k-2 were confirmed to confer P. sojae 3

resistance by soybean transformation (Gao et al. 2005). 4

Genetic analysis of P. sojae has revealed at least 12 single avirulence genes 5

matching Rps genes (Whisson et al. 1994, 1995; Tyler et al. 1995; Gijzen et al. 1996; 6

May et al. 2002). Several pairs of avirulence genes such as Avr1b and Avr1k (Whisson et 7

al. 1995; Shan et al. 2004), Avr3a and Avr5 (Whisson et al. 1995), and Avr4 and Avr6 8

(Whisson et al. 1995; Gijzen et al. 1996), appeared closely linked. Two of these pairs 9

have proven to correspond to a single molecular gene, namely Avr3a and Avr5 (Qutob et 10

al. 2009; Dong et al. 2011b), and Avr4 and Avr6 (Dou et al. 2010). In addition to Avr3a/5 11

and Avr4/6, Avr1a (Qutob et al. 2009), Avr1b-1 (Shan et al. 2004), Avr3b (Dong et al. 12

2011a) and Avr3c (Dong et al. 2009) have been cloned and characterized. The first cloned 13

avirulence gene, Avr1b-1 (Shan et al. 2004), encodes an effector protein that has an N-14

terminal host-targeting domain with motifs (known as RxLR and dEER) found in all 15

cloned P. sojae avirulence effectors. The RxLR-dEER domain mediates effector 16

translocation into host cells in the absence of pathogen machinery, and entry involves 17

binding to phosphatidyinositol-3-phosphate (PI3P) on the surface of the plant plasma cell 18

membrane (Dou et al. 2008a; Kale et al. 2010; Sun et al. 2012). The C-terminal 19

sequences of Avr1b are moderately conserved and have the ability to suppress host 20

defense (Dou et al. 2008b). 21

Oomycetes including P. sojae, P. infestans and Hyaloperonospora arabidopsidis 22

evade recognition by matching host R gene(s) through mutations in the responsible 23

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avirulence genes (substitutions and deletions) and/or through transcriptional silencing of 1

those genes (Qutob et al. 2013; reviewed by Tyler, 2009). Examples in P. sojae include 2

Avr1a, Avr1b and Avr3a/5 (Shan et al. 2004; Qutob et al. 2009; Dong et al. 2011b). 3

Although eight of the 12 genetically identified P. sojae avirulence genes have so far been 4

cloned, the avirulence gene(s) corresponding to Rps1k have still not been clearly 5

elucidated. Here, we demonstrate that two genetically closely-linked but distinct RxLR 6

effector genes, including Avr1b-1 and a new gene, Avr1k, are responsible for Rps1k-7

mediated gene-for-gene resistance. The recognition of two distinct effectors by plants 8

carrying the Rps1k allele suggests a possible molecular basis for the durability of this 9

resistance gene. 10

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RESULTS 12

Genetic characterization of the Avr1b/Avr1k locus 13

Previously, Avr1b-1 and Avr1k were genetically defined as single dominant genes 14

that co-segregated in all F2 progeny of the cross between isolates of race 7 (Avr1b+, 15

Avr1k+) and race 25 (Avr1b–, Avr1k–) (Shan et al. 2004; Tyler, 1995;Whisson et al. 16

1995). Avr1b-1 was genetically mapped to a single 60-kb BAC, 3E16, leading to its 17

cloning (Shan et al. 2004). Avr1k was also localized to the same BAC by characterization 18

of F2 and F3 progeny from the cross P6497 (race 2, Avr1b–, Avr1k+)×P7076 (race 19, 19

Avr1b–, Avr1k–) and F2 progeny of the cross P7064 (race 7, Avr1b+, Avr1k+)×P7081 20

(race 25, Avr1b–, Avr1k–) (Shan et al. 2004). As part of that study, the region surrounding 21

Avr1b-1 was sequenced from P6497, P7064 and P7076. When the complete superfamily 22

of RxLR genes was identified through use of a hidden markov model (Jiang et al. 2008), 23

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a second RxLR effector gene, Avh331, was discovered 5 kb from Avr1b-1 (Fig. 1A,B). 1

Inspection of the sequence of Avh331 in virulent strains such as P7076 revealed a 2

frameshift mutation in the gene in each strain, suggesting that it might be recognized by 3

Rps1k (Fig. 1B,C). On the other hand, some avirulent strains such as P7063 (race 6) and 4

P7064 (race 7) also carried the frameshift mutation in Avh331. Since P7063 and P7064 5

carried functional Avr1b-1 genes, and no strains could ever be isolated that were virulent 6

on Rps1k but avirulent on Rps1b (Whisson et al. 1995; Shan et al. 2004), we 7

hypothesized that Avr1b, as well as Avh331, might be recognized by Rps1k. This 8

hypothesis was encouraged by the observation that purified recombinant Avr1b protein 9

from Pichia pastoris could trigger a small amount of localized necrosis at the site of 10

infiltration of soybean leaves carrying Rps1k (Shan et al. 2004) and that Avh331 protein, 11

purified after expression in E. coli, could trigger cell death on soybean plants containing 12

Rps1k (Kale et al. 2010). For simplicity, we refer to Avh331 as Avr1k in the remainder of 13

the paper. 14

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Avr1b confers avirulence in the presence of Rps1k 16

To test the hypothesis that plants carrying Rps1k recognize Avr1b, we used 17

particle bombardment experiments with the Avr1b-1 gene together with a gene encoding 18

beta-glucuronidase (GUS) in soybean leaves to measure cell death triggered by Avr1b in 19

the presence of Rps1k. We used a double-barreled attachment for the Bio-Rad Gene Gun 20

that enables simultaneous bombardment of a DNA test sample and a control sample into 21

two sides of a soybean leaf with the same shot, which greatly reduces the variance of the 22

results (Dou et al. 2008a,b; Kale et al. 2010; Gu et al. 2011; Liu et al. 2011). Fig. 2 shows 23

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that the number of tissue patches staining blue as a result of GUS expression was reduced 1

around 76% by Avr1b-1 expression compared to the empty vector (EV) in leaves 2

containing Rps1k (Haro15) but not in leaves lacking Rps1k (rps) (p < 0.001). A similar 3

reduction was observed in the presence of Rps1b (Fig. 2) (Dou et al. 2008a). These 4

results indicate that Avr1b-1 expression triggers Rps1k-dependent cell death in soybean 5

leaves, as well as Rps1b-dependent cell death. 6

In order to test in vivo whether Avr1b-1 confers specific avirulence on P. sojae in 7

the presence of Rps1b or Rps1k, we examined the phenotypes of two stably transformed 8

P. sojae lines (T17 and T20) that produce high levels of Avr1b-1 mRNA (Dou et al. 9

2008a). The transformants were avirulent on soybean cultivars containing Rps1b (Haro13) 10

or Rps1k (Haro15) in a hypocotyl inoculation assay compared with plants lacking either 11

Rps gene (Fig. 3B; Table 1), whereas the untransformed recipient strain (P7076) was 12

virulent on all the cultivars. We also measured their virulence phenotypes quantitatively 13

using a root lesion progression assay. The lesion lengths were significantly (p < 0.001) 14

reduced by 2-3 fold on soybean roots carrying Rps1k or Rps1b, compared with roots 15

lacking the Rps genes (Fig. 3A). 16

Together these results indicate that the Avr1b gene product is recognized by plant 17

tissue containing either Rps1k or Rps1b when Avr1b-1 is overexpressed in either the plant 18

or the pathogen. 19

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Silencing of Avr1b-1 results in loss of the avirulence phenotype on soybean cultivars 21

containing Rps1k or Rps1b 22

In order to confirm that the endogenous Avr1b-1 gene was responsible for 23

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avirulence on Rps1k cultivars as well as Rps1b cultivars, and to rule out the possibility 1

that recognition by Rps1k plants was caused by over-expression of Avr1b-1, we silenced 2

Avr1b-1 in isolate P7063 (race 6) that is avirulent on soybeans containing Rps1b or Rps1k. 3

P7063 contains a frameshift mutation in Avr1k, therefore avirulence on Rps1b or Rps1k 4

plants should be due to the Avr1b-1 gene alone in this isolate. Three plasmids with sense, 5

antisense and hairpin Avr1b-1 constructs (Supplementary Fig. S2) driven by the 6

constitutive Ham34 promoter (Judelson et al. 1991) were used to produce stable 7

transformants of P7063 (McLeod et al. 2008; Dou et al. 2008a); all three strategies for 8

silencing Phytophthora genes have been successfully used previously (Ah-Fong et al. 9

2008). Three independent transformants (T5-15 from hairpin, T7-1 from antisense and 10

T8-5 from sense constructs, respectively) with reduced Avr1b-1 expression were obtained. 11

Integration of the constructs into the genome of the transformants was confirmed by 12

Southern blot analysis (Fig. 4A). The transcript of Avr1b-1 was almost undetectable in 13

Northern blots while it was readily detectable in the wild type (Fig. 4B). To determine 14

whether avirulence in the presence of Rps1b or Rps1k correlated with Avr1b-1 mRNA 15

level, we inoculated the three silenced transformants onto hypocotyls of soybean cultivars 16

containing Rps1b (Haro13) and Rps1k (Haro15). All three silenced transformants could 17

kill soybean seedlings containing Rps1b or Rps1k (Figure 4C; Table 1). In contrast, the 18

control GUS transformant T9-1 remained avirulent on the cultivars containing Rps1b or 19

Rps1k. These results indicate that the Avr1b gene product is responsible for avirulence in 20

the presence of Rps1k as well as Rps1b in isolates like P7063 that lack a functional Avr1k 21

gene. 22

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Characterization of Avr1k 1

We used real time RT-PCR to measure the transcript levels of Avr1k in P6497 in 2

different infection stages. The actin gene was used as a reference. The transcript levels of 3

Avr1k in the cultured mycelium were low but detectable. Avr1k transcript levels peaked at 4

12 h post-inoculation at levels 20-fold above the mycelial level (Fig. 5), consistent with 5

the recognition of the Avr1k gene product during infection. 6

Kale et al (2010) previously showed that particle-bombardment-mediated 7

transient expression of Avr1k (called Avh331 in that paper) in soybean leaves conferred 8

Rps1k-dependent cell death (Fig. 2). Transient expression of Avr1k in Rps1b-containing 9

leaves did not however trigger cell death (Fig. 2) indicating that Rps1b-containing tissue 10

can recognize Avr1b but not Avr1k. This result is consistent with the observation that 11

isolate P6497, that expresses Avr1k but not Avr1b-1, is virulent on Rps1b-containing 12

cultivars, but is avirulent on Rps1k-containing cultivars. 13

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Avr1k confers avirulence in the presence of Rps1k 15

To determine directly if Avr1k could confer avirulence during infection, the Avr1k 16

gene was fused with a C-terminal His tag and placed under control of a strong 17

constitutive promoter rpL41 (Dou et al. 2008a), and then introduced into a strain, P7076, 18

that is virulent on cultivars carrying the Rps1k gene. Only one Avr1k-expressing stable 19

transformant (T12) was obtained from more than one hundred putative transgenic lines 20

that could grow on the G418-selection medium (Fig. 6A,B). Over-production of Avr1k 21

transcripts and of the Avr1k protein product were validated by RT-PCR and Western blot 22

analyses, respectively (Fig. 6B,C). Neither the transcript nor protein product could be 23

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detected in the recipient P7076 (Fig. 6B,C). We inoculated T12 and the wild type onto 1

hypocotyls of a cultivar containing Rps1k (Williams 82) and a control isoline Williams 2

(rps). When Rps1k was present, T12 could not kill the soybean seedlings while the wild 3

type could (Fig. 6D; Table 1). Both T12 and the wild type were virulent on Williams (Fig. 4

6D, Table 1.) These results indicated that Avr1k could confer avirulence on Rps1k-5

containing soybean cultivars, supporting the hypothesis that Avr1k can be recognized by 6

Rps1k gene products. 7

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Silencing of Avr1k results in loss of the avirulence phenotype on soybean cultivar 9

containing Rps1k 10

To directly test whether Avr1k was responsible for the avirulence of P. sojae 11

isolates in the presence of Rps1k, and to rule out the possibility that recognition of Avr1k 12

by Rps1k plants is an artifact of over-expression, we silenced Avr1k in P6497. The same 13

construct used for Avr1k over-expression was used for co-transformation together with a 14

plasmid containing the G418-resistance selectable marker; the sense construct can be as 15

effective in silencing target genes as antisense and hairpin constructs (Fig. 4) (van West 16

et al. 1999; Ah-Fong et al. 2008). We obtained more than one hundred putative 17

transformants that could grow on the G418-selection medium. The transformed lines 18

were screened at 12 h post-inoculation for loss of Avr1k transcript accumulation. A total 19

of three independent transformants, T30, T51 and T131, were obtained with reduced 20

Avr1k transcript levels, less than 20% relative to the wild type as measured by qRT-PCR 21

(Fig. 7A,B). We then examined the virulence phenotype of the silenced transformants on 22

soybean by hypocotyl inoculation. All three silenced transformants could kill Rps1k-23

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containing soybean seedlings (Fig. 7C, Table 1). In contrast, the recipient P. sojae isolate 1

P6497 and a transformed line T32, in which Avr1k was not silenced, remained avirulent 2

on cultivars containing Rps1k (Fig. 7C, Table 1). These results confirm that the 3

endogenous Avr1k gene is responsible for Rps1k-mediated “gene-for-gene” resistance in 4

P. sojae isolates lacking Avr1b-1 expression. 5

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Sequence polymorphisms in Avr1k 7

To more broadly survey polymorphisms in Avr1k that may be associated with the 8

loss of the avirulence phenotype in virulent races, we cloned this gene from eight P. sojae 9

isolates, P6954 (Race 1, phenotype Avr1b+, Avr1k+), P6497 (Race 2, Avr1b–, Avr1k+), 10

P7063 (Race 6, Avr1b+, Avr1k+), P7064 (Race 7, Avr1b+, Avr1k+), P7360 (Race11, 11

Avr1b–, Avr1k+), P7076 (Race 19, Avr1b–, Avr1k–), P7081 (Race 25, Avr1b–, Avr1k–) 12

and PT2004C2.S1 (Race 30, Avr1b–, Avr1k–) (Forster et al. 1994; Zhou et al., 2009). 13

Three isolates, P6954, P6497 and P7360, contained identical Avr1k gene sequences with 14

a full open reading frame, and all were avirulent on cultivars carrying Rps1k (Table 2). 15

The other five isolates exhibited a variety of polymorphisms relative to Avr1k from the 16

three avirulent isolates, but shared a common insertion of eight nucleotides of 17

“TGCTACTT” between the regions encoding for the signal peptide and RxLR motif of 18

Avr1k, leading to a frameshift and early stop in the open reading frame (Fig. 1, 19

Supplementary Fig. S3). These isolates were all virulent on cultivars containing Rps1k, 20

except for isolates expressing a functional Avr1b-1 gene, namely P7063 and P7064 21

(Table 2). Overall the relationship between avirulence on Rps1k plants and 22

polymorphisms in Avr1b-1 and Avr1k supports the conclusion that Rps1k gene products 23

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can recognize both Avr1b and Avr1k proteins. 1

2

DISCUSSION 3 4 We have demonstrated that two closely linked RxLR effector genes, Avr1b-1 and 5

Avr1k confer avirulence in P. sojae infecting soybean cultivars containing Rps1k. Over-6

expression of either gene in P. sojae transformants conferred specific avirulence, while 7

silencing of either gene in appropriate P. sojae strains eliminated avirulence. Only P. 8

sojae isolates carrying mutations in both Avr1b-1 and Avr1k could infect cultivars 9

carrying Rps1k. Rps1k has provided durable resistance against P. sojae in the northern 10

mid-west of the United States since the 1970’s. Only in recent years has Rps1k-mediated 11

resistance become less reliable as P. sojae strains lacking functional Avr1k and Avr1b-1 12

genes have spread. It seems likely that the durability of Rps1k has resulted from its ability 13

to confer recognition of two different RxLR effectors. The very small physical distance 14

of 5 kb between Avr1b-1 and Avr1k would have greatly reduced genetic recombination 15

events that might have enabled mutations in Avr1b-1 and Avr1k to be combined through 16

sexual outcrossing (Forster et al. 1994). 17

P. sojae has evolved several distinct mechanisms to avoid the recognition of 18

avirulence effectors by soybean R proteins. Alterations of the Avr1b-1 gene that result in 19

loss of recognition by Rps1b cultivars include amino acid substitutions, complete 20

deletions of the gene, or loss of transcript accumulation (Shan et al. 2004; Cui et al. 2012). 21

In the case of Avr1k, all alleles found in isolates not recognized by Rps1k cultivars 22

contained the same insertion of eight nucleotides “TGCTACTT” in the coding region, 23

producing a pseudogene. The origin of this sequence is unclear; it is not a duplication of a 24

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nearby sequence for example. 1

Avr1b represents the third example in which a P. sojae effector is recognized by 2

products of two distinct soybean resistance genes (Rps1b and Rps1k). In the case of 3

Avr4/6, distinct domains of the effector protein were recognized by plants containing the 4

two relevant R genes, Rps4 and Rps6, (Dou et al, 2010). In the case of Avr3a/5, distinct 5

sets of Avr3a/5 alleles were recognized by plants containing the two relevant R genes, 6

Rps3a and Rps5. In the case of Avr1b however, we were unable to identify any mutants 7

or alleles of the effector that were differentially recognized by plants containing Rps1k 8

and Rps1b. The two R genes are distinguished only by their ability to confer recognition 9

of Avr1k. Rps1b and Rps1k represent two of six alleles of the Rps1 locus. The Rps1 10

locus is highly polymorphic and contains a large number of similar genes encoding CC-11

NB-LRR proteins (Bhattacharyya et al. 2005; Gao et al. 2008). Two near-identical genes 12

conferring P. sojae resistance were cloned from the Rps1k locus, Rps1k-1 and Rps1k-2 13

(Bhattacharyya et al. 2005; Gao et al. 2005, 2008). However it is currently unclear 14

whether each individual cloned gene confers recognition of both Avr1b and Avr1k, 15

which have very different amino acid sequences. It is possible that different CC-NBS-16

LRR proteins encoded by genes within the broader Rps1k region recognize the two 17

different effectors. Furthermore, it is possible that the Rps1b and Rps1k loci share one or 18

more identical genes that encode R proteins able to recognize Avr1b. In Arabidopsis 19

thaliana, the R protein Rpm1 can recognize two dissimilar avirulence genes from 20

Pseudomonas syringae, AvrB and AvrRpm1 (Bisgrove et al. 1994). However in that case, 21

Rpm1 guards a plant protein, RIN4, that is targeted by the two bacterial effectors 22

(Mackey et al. 2002). Similarly, in tomato the Cf2 resistance gene can recognize the 23

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distinct effectors Avr2 from the fungus Cladosporium fulvum and Gr-VAP1 from the 1

potato cyst nematode Globodera rostochiensis because both effectors target a common 2

plant protein, Rcr3, guarded by Cf2 (Lozano-Torres et al. 2012). In flax rust Melampsora 3

lini, the AvrL567-A gene product is targeted by three allelic R gene products L5, L6 and 4

L7 (Catanzariti et al. 2006). In that case, the R proteins interact directly with the effector 5

(Dodds et al. 2006). 6

Like most of the avirulence genes cloned from oomycetes, including Avr1b-1, 7

Avr1k encodes a protein with the cell entry motifs, RxLR and dEER. Accordingly, 8

synthesis of Avr1k lacking its secretory leader inside soybean cells following 9

bombardment of leaves with an Avr1k gene produced cell death specifically in leaves 10

carrying Rps1k (Kale et al. 2010), indicating that Avr1k interacts with Rps1k gene 11

products inside soybean cells. Kale et al (2010) showed that purified Avr1k protein 12

could enter both root and leaf cells of soybean autonomously; in both cases the RxLR and 13

dEER motifs of Avr1k were required. Kale et al (2010) further showed that Avr1k could 14

bind PI3P and that binding, which required the RxLR and dEER motifs, was required for 15

cell entry. 16

The C-terminal domain of Avr1k contains two modules consisting of W-Y motifs, 17

one of which also includes an L motif (Jiang et al. 2008; Dou et al. 2008a) (Fig. 1B). 18

These motifs occur in almost half of the identified P. sojae RxLR effectors (Jiang et al. 19

2008; Dou et al. 2008a) and structural studies have revealed that W-Y modules form a 20

structural unit that supports a diversity of solvent-exposed surfaces (Boutemy et al. 2011). 21

Both Avr1b-1 and Avr1k are up-regulated during infection (Shan et al., 2004; 22

Wang et al. 2011; this study), and both can suppress cell death triggered by the PAMP 23

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INF1, by other P. sojae effectors, or even by the mouse pro-apoptotic protein Bax (Dou 1

et al. 2008b; Wang et al. 2011). Avr1k could also suppress mitogen-activated protein 2

kinase (MAPK)-based plant defense (Cheng et al. 2012). The plant proteins targeted by 3

Avr1b and Avr1k are currently unknown. P. infestans effector Avr3a, which is closely 4

related to Avr1b (Armstrong et al. 2005), suppresses cell death by targeting and then 5

stabilizing host U-box E3 ligase CMPG1 (Bos et al. 2010). Since both Avr1b and Avr1k 6

can suppress cell death triggered by Bax, it is possible that the two effectors interact with 7

fundamental components of the plant cell death machinery. Identification of the plant 8

targets of Avr1b and Avr1k should reveal how these two proteins can suppress a diversity 9

of plant immune functions, and may shed light on their interactions with the products of 10

Rps1k and Rps1b genes. 11

12

MATERIALS AND METHODS 13

14

Bioinformatic Analyses 15

The P. sojae and P. ramorum genome sequences and predicted gene models were 16

accessed at eumicrobedb.org. Functional analyses of predicted genes were based on 17

BLAST and FASTA searches against public databases. Sequence similarities were 18

determined using the web site http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi. 19

Genes sharing best bidirectional BLAST matches were considered to be candidate 20

ortholog pairs. Synteny maps among the genomes of Phytophthora species were 21

generated by using PHRINGE (Dehal and Boore, 2006). Identification of transposable 22

elements in syntenic regions were carried out with the program CRNSOR 23

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(www.girinst.org/censor/index.php) (Kohany et al. 2006) by searching Repbase 1

( http://www.girinst.org/repbase/index.html) (Jurka et al. 2005). 2

P. sojae strains and manipulation 3

Phytophthora sojae isolates, P6954 (Race 1), P6497 (Race 2), P7063 (Race 6), 4

P7064 (Race 7), P7360 (Race 11), P7074 (Race 17), P7076 (Race 19), P7081 (Race 25), 5

P9073 (Race 25) and Race 30 (Forster et al. 1994; Whisson et al. 1995; Zhou et al. 2009) 6

were routinely grown and maintained on V8 agar at 25°C in the dark. Procedures for P. 7

sojae transformation, screening of P. sojae transformants and identification of Avr1k 8

polymorphisms were as described (Dou et al. 2008a,b). 9

10

Transcriptional profiling 11

To characterize Avr1k expression during infection, total RNA of P. sojae P6497 12

was collected from mycelium sandwiched between pairs of soybean leaves (Chen et al. 13

2007). The mycelium was removed at 2 hours after inoculation (hpi), 4 hpi and 8 hpi, or 14

the infected soybean leaves were collected at 12 hpi, 24 hpi and 36 hpi. All of the 15

collected samples were immediately frozen in liquid nitrogen and stored at -80°C until 16

RNA isolation. RNAsimple Total RNA Kit (TIANGEN) was used for total RNA 17

extraction and SYBR green real-time RT-PCR assays (TaKaRa) were carried out for 18

Avr1k transcript profiling analysis following the manufacturer’s described methods. We 19

used Avr1k-RT-F and Avr1k-RT-R primers for Avr1k, and actinAF2 and actinAR2 20

primers for the reference gene, P. sojae actin gene (all primers are listed in 21

Supplementary Table S2). The reaction conditions were as follows: 95°C for 30 s, 40 22

cycles of (95°C for 5 s, and 60°C for 34 s); followed by 95°C for 15 s, 60°C for 1 min, 23

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and 95°C for 15 s to obtain melt curves. The Ct of each gene relative to average Ct values 1

of the housekeeping gene (Ct=Ctgene-CtHKaverage ) were determined and analyzed using 2

ABI 7500 System Sequence Detection Software Version 1.4 (Schuhmacher et al. 2010). 3

4

Construction of Plasmids 5

P. sojae transformation plasmids, pUN (NptII gene driven by P. sojae rpL41 6

promoter) and pHamAvr1b, plant transient expression plasmids pUCAvr1b and pUCGUS 7

were described by Dou et al. (2008a). To make the anti-sense construct of Avr1b-1, the 8

amplicons of Avr1b-1 produced with primers of Avr1bF and Avr1bR were digested with 9

Kpn I and inserted into Kpn I-digested pHamT35 (Judelson et al. 1991). The antisense 10

construct of Avr1b-1 was identified by restriction enzyme digestion. The hairpin 11

construct of Avr1b-1 [pHam:Avr1b(anti-sense):rpL41 intron: Avr1b (sense)] was obtained 12

by two-step PCR with the primers UblIF, UblIR, Avr1bHPF and Avr1bHPR and ligation 13

of three resultant fragments. To make the Avr1k construct for P. sojae transformation 14

(pHam34-Avr1k), the Avr1k gene was amplified with the primers Avr1k-Sma I-F, Avr1K-15

His-KpnI-R1 and Avr1K-His-KpnI-R2, digested with Sma I and Kpn I, and then inserted 16

into the vector pUN digested with the same enzymes. All the above constructs were 17

confirmed by sequencing. All constructs for over-expressing Avr1b-1 and Avr1k utilized 18

the full length coding region including the secretory leader. 19

20

Characterization of P. sojae transformants 21

The two transformants (T17 and T20) that over-express Avr1b-1 were previously 22

described by Dou et al. (2008a). Transformants with reduced Avr1b-1 transcript levels 23

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were first screened following the methods of Dou et al. (2008a). For DNA gel blot 1

hybridization, we purified genomic DNA from mycelium as described (Judelson et al. 2

1991) then digested 10 µg genomic DNA with Kpn I or Eco RI+Hin dIII. The digested 3

DNA was size fractionated on a 0.7% agarose gel followed by transfer to Hybond Nt 4

nylon membrane (Amersham). For RNA gel blot analysis, total RNA from the 5

transformants was isolated using the RNeasy plant mini kit (Qiagen) according to the 6

manufacturer’s recommendations. We electrophoresed samples of 5 to 10 µg total RNA 7

in a 1% agarose gel in MOPS running buffer at 45 mV for 50 min. The gel was then 8

stained with an RNA staining buffer for 15 min. The gel was transferred overnight with 9

20×SSC buffer. DNA probes for Avr1b-1 were synthesized using a PCR digoxigenin 10

probe synthesis kit according to the manufacturer’s manual (Roche Diagnostics). The 11

membrane was hybridized, and chemiluminescent detection was performed following the 12

kit instructions. 13

Putative P. sojae transformants containing Avr1k were screened in three steps. 14

First, DNA from each line was amplified with oligonucleotides Avr1k-Sma I-F and 15

Ham34-R, which are primers for the 5’ end of Avr1k and for the Ham34 terminator, 16

respectively. Next, RT-PCR was performed on RNA extracted from each line using 17

oligonucleotides Avr1k-harf-F and Avr1k-harf-R to confirm Avr1k expression. Finally, 18

the transcript levels derived from the Avr1k transgenes were accurately quantitated by 19

real-time PCR as described above. Since the transcript levels of Avr1k are almost un-20

detectable in mycelia and highly induced at 12 hpi, Avr1k silencing transformants were 21

screened at 12 hpi. 22

For over-expression transformants of Avr1k, Western blots were used to confirm 23

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the protein levels. Total mycelial protein was extracted using Plant Protein Miniprep Kit 1

(TIANDZ) for Western blot assays. A standard sodium dodecyl sulfate polyacrylamide 2

gel electrophoresis (SDS-PAGE) protocol was used for protein separation. Proteins were 3

transferred onto polyvinylidene difluoride (PVDF) membranes using a semi-wet 4

apparatus (Bio-Rad) according to the product instructions. Western blotting was 5

performed as described (Yu et al. 2012). Anti-His-tag monoclonal antibody (Sigma-6

Aldrich) and anti-mouse IgG-peroxidase conjugate (Sigma-Aldrich) were used as the 7

primary and secondary antibodies, respectively. The membrane was treated with 8

Chemiluminescent Peroxidase Substrate-1 (Thermo Scientific) for 5 min. The membrane 9

was briefly drained and exposed to BioMax (Kodak, USA) light film for several different 10

time periods (depending on exposure). 11

The avirulence phenotypes of selected transformants were evaluated by hypocotyl 12

inoculation (Tyler et al. 1995) using soybean cultivars Williams (rps), Williams 82 13

(Williams background, Rps1k), Haro(1-7) (rps), Haro13 (Harosoy background, Rps1b) 14

and Haro15 (Harosoy background, Rps1k). The plants were grown in the greenhouse with 15

a 16-hour photoperiod at 25°C. One to two days after the first primary leaf appeared, the 16

hypocotyl of the soybean was wounded with a short incision and the incision was 17

inoculated with a small piece of V8 agar cut from the edge of a 3-d-old colony. 18

Thereafter, the plants were incubated in the greenhouse under the conditions described 19

above. The numbers of dead and surviving plants were counted 4 d after inoculation. The 20

numbers of surviving plants from rps and Rps1k or Rps1b cultivars were compared using 21

Fisher’s exact test. Only the transformants producing significant differences between rps 22

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and Rps1k or Rps1b cultivars were classified as avirulent. Each avirulence or virulence 1

determination was repeated at least three times. 2

3

Particle bombardment assays for avirulence and virulence phenotypes 4

Particle bombardment assays were carried out using a double-barreled extension 5

of the Bio-Rad He/1000 Particle Delivery System (Dou et al. 2008b). To quantitate the 6

avirulence (R gene-dependent cell death) conferred by Avr1b-1 constructs, DNA carrying 7

the constructs (1.7 µg per shot) was co-bombarded into soybean leaves along with DNA 8

carrying a ß-glucuronidase (GUS) reporter gene (1.7 µg per shot). Avirulence activity 9

was measured as the reduction in the number of blue-staining GUS-positive spots in 10

leaves carrying Rps1k compared to leaves lacking either resistance gene. The double-11

barreled device was used to deliver a parallel control shot in every case that contained 12

GUS DNA plus empty vector DNA. For each pair of shots, the logarithm of the ratio of 13

the number of blue spots with various Avr1b-1 constructs to the number with the empty 14

vector control was calculated. Each assay consisted of eight pairs of shots and was 15

conducted at least twice. The log-ratios from all the Rps1k leaves were then compared to 16

those from the non-Rps1k leaves using the Wilcoxon rank sum test. 17

18

Acknowledgements 19

This work was supported by funds to D.D. from the National Natural Science 20

Foundation of China (30971889), the Natural Science Foundation of Jiangsu Province 21

(BK2012027) and the Key Project of the Chinese Ministry of Education (E200909), and 22

grants to B.M.T. from the Agriculture and Food Research Initiative of the USDA 23

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21

National Institute for Food and Agriculture numbers 2001-35319-14251, 2002-35600-1

12747, 2007-35319-18100, and 2007-35600-18530, and from the U.S. National Science 2

Foundation, numbers MCB-0242131, MCB-0731969 and IOS-0924861. 3

4

Author contributions 5

The work was planned by B.M.T., D.D., S.D.K. and Y.W. The experiments were 6

carried out by T.S., S.D.K., F.D.A., L.S., L.L. and D.D. Bioinformatics was carried out 7

by D.S. The paper was written by D.D. and B.M.T. with input from all authors. 8

9

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1

Table 1. Characterization of P. sojae stable transformants 2 3

Strains a Transgene

b

Avr

Transcripts c

Virulence d

p value e

rps Rps1k

P7076 (Avr1b–, Avr1k

–) none none 42/44 V 17/19 V n.s.

T17 f Avr1b-OX Avr1b ++ 34/35 V 1/22 A <0.001

T20 f Avr1b-OX Avr1b ++ 26/28 V 1/16 A <0.001

T12 Avr1k-OX Avr1k ++ 27/33 V 0/33 A <0.001

P7063 (Avr1b+, Avr1k

–) none Avr1b + 25/26 V 2/25 A <0.001

T5-17 Avr1b-SIL Avr1b – 27/30 V 20/26 V n.s.



T9-1 GUS Avr1b + 26/32 V 1/17 A <0.001

P6497 (Avr1b–, Avr1k

+) none Avr1k + 32/32 V 1/50 A <0.001

T30 Avr1k-SIL Avr1k – 38/38 V 39/48 V n.s.



T32 NptII only Avr1k + 15/15 V 1/36 A <0.001

4 a Recipient strains and their genotypes are listed above the transformants derived from 5

them. 6

b The presence of transgenes in the transformants was verified by PCR and/or Southern 7

blots as indicated in Figures 4, 6 & 7. OX = overexpression transgene present; SIL = 8

silencing transgene present. Transgene in control transformant T9 consisted of nptII plus 9

a beta-glucuronidase gene; transgene in T32 consisted of nptII plus a promoter-terminator 10

construct lacking any coding region. 11

c Transcript levels of the indicated genes, as determined by Northern blots, qualitative 12

RT-PCR and/or by quantitative RT-PCR (qRT-PCR) as indicated in Figures 4, 6, and 7. – 13

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= reduced or undetectable transcript levels; + = native transcript levels; ++ = elevated 1

transcript levels 2

d The virulence of each strain was tested by inoculation of seedlings containing Rps1k 3

(Williams 82 or HARO15) or no rps gene (Williams or HARO(1-7)1) as described in the 4

Materials and Methods. The number of dead seedlings/total inoculated seedlings is 5

shown, summed from all replicates. A = Avirulent; V = Virulent. 6

e Fisher’s exact test (one tailed) was used to compare the frequency of seedling survival 7

between rps and Rps1k plants. A significant p value (<0.001) indicates that the 8

transformant’s phenotype is avirulent. n.s. = not significant. 9

f T17 and T20 were described by Dou et al. (2008a) including transgene verification, 10

Avr1b-1 transcript levels and Avr1b phenotype.11

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Table 2. Avr1b and Avr1k Genotypes and Phenotypes 1

2

a Avirulence (A) or specific virulence (V) in the presence of the indicated R gene 3

b Avr = Avirulence allele; Vir = Virulence allele; nt = no transcript; fs = frameshift 4

c The contribution of each gene, Avr1b-1 or Avr1k, to recognition by Rps1k plants + = 5

positive contribution; – = no contribution; ND = not determined 6

7

Race Isolate Phenotypea Avirulence gene alleleb Avr1k phenotype

contributionc Rps1b Rps1k Avr1b Avr1k

Race 1 P6954 A A Avr Avr Avr1b+ Avr1k+

Race 2 P6497 V A Vir(nt) Avr Avr1b– Avr1k+

Race 6 P7063 A A Avr Vir (fs) Avr1b+ Avr1k–

Race 7 P7064 A A Avr Vir (fs) Avr1b+ Avr1k–

Race 11 P7360 V A ND Avr Avr1b(ND) Avr1k+

Race 19 P7076 V V Vir Vir (fs) Avr1b– Avr1k–

Race 25 P7081 V V Vir Vir (fs) Avr1b– Avr1k–

Race 30 PT2004C2.S1 V V ND Vir (fs) Avr1b– Avr1k–

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Figure legends 1

2

Fig. 1. Identification of the RxLR effector gene Avr1k 3

A, Region of P. sojae scaffold_6 (3070060-3070120; version 5 of the genome sequence) 4

containing Avr1b-1 and Avr1k. Black arrows indicate genes with syntenic orthologs in 5

other Phytophthora species, and white arrows show genes with no orthologs. Further 6

details regarding the genes in this region are given in Supplementary Fig. S1, and 7

Supplementary Table S1. B, predicted amino acid sequence encoded by Avr1k in the 8

genome sequence of P6497, with key sequence features underlined. The triangle 9

indicates the 8 bp frameshift insertion in some alleles. C, alleles of Avr1k in other 10

isolates; unlabeled black bars indicate the positions of nucleotide substitutions, the 8 bp 11

frameshift insertion and the TGA stop codon it leads to are labeled. Nucleotide sequence 12

alignment of the alleles is shown in Supplementary Fig. S2. 13

14

Fig. 2. Cell death responses triggered by Avr1b-1 and Avr1k in the presence of Rps1b 15

and Rps1k. Responses were measured by double-barreled particle bombardment of 16

soybean leaves (Dou et al. 2008b; Kale et al. 2010), in which a mixture of GUS plus Avr 17

gene DNA was bombarded through one barrel and GUS plus empty vector DNA was 18

bombarded through the second barrel. Cell death results in a lowered ratio of GUS-19

positive tissue patches between Avr gene and control DNA bombardments. A, Average 20

cell death ratios from 14-16 paired bombardments each. Error bars indicate standard 21

errors. Asterisks indicate a significant difference (p < 0.001; Wilcoxon rank sum test) 22

between bombardment of Rps gene-containing leaves (Rps1b: L77-1879 or Haro13; 23

Rps1k: Williams 82 or Haro15) compared to non-Rps containing (rps: Haro(1-7)1 or 24

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Willaims) leaves. B, Illustrative examples of double barrel bombardments. Left barrels 1

contained GUS plasmid plus empty vector plasmid. Right barrels contained GUS 2

plasmid plus Avr1b-1 driven by 35S promoter. 3

Fig. 3. Overexpression of Avr1b-1 in P. sojae produces an avirulence phenotype on 4

soybean cultivars containing Rps1k or Rps1b. A, root lesion lengths 5 d after inoculation. 5

Lesions on the roots of 30 seedlings inoculated with wild-type or Avr1b-overexpressing 6

P. sojae lines were measured in each of five independent experiments. Error bars show 7

standard error. P values were determined with a t test. B, representative phenotypes in 8

hypocotyl inoculation assays of wild type and transgenic lines on soybean cultivars: 9

Haro1 (rps, Harosoy background), Haro13 (Rps1b, Harosoy background) and Haro15 10

(Rps1k, Harosoy background). Photographs were taken 4 days after inoculation. T20 11

produced similar results to T17. Quantitative analysis of seedling killing by all lines is 12

presented in Table 1. 13

Fig. 4. Gene silencing of Avr1b-1 produces a virulence phenotype on soybean lines 14

containing Rps1b or Rps1k. A, DNA gel blots of transgenic P. sojae hybridized with an 15

Avr1b probe. T5-17, T7-1and T8-5 are three transformants with different constructs. 16

Control line T9-1 was transformed with a GUS construct. H/E = HindIII+EcoR1 double 17

digest; K = KpnI digest. Arrows indicate fragments corresponding to the endogenous 18

Avr1b-1 gene. B, RNA gel blots of RNA extracted from P. sojae hyphae grown in vitro. 19

Probe was Avr1b-1. The bottom panel shows ethidium bromide staining of RNA prior to 20

transfer. 21

C, representative phenotypes in hypocotyl inoculation assays of wild type and transgenic 22

lines on soybean cultivars: Haro52 (Rps5, Harosoy background), Haro13 (Rps1b, 23

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Harosoy background) and Haro15 (Rps1k, Harosoy background). Photographs were 1

taken 4 days after inoculation. T8-5 produced similar results to T5-15 and T7-1. 2

Quantitative analysis of seedling killing by all lines is presented in Table 1. 3

4

Fig. 5. Avr1k transcript levels during infection measured by quantitative real-time RT-5

PCR. Total RNA was extracted from mycelia (0 h), and P6497-infected soybean leaves 6

at 2 to 36 h post-inoculation. Susceptible soybean cultivar Williams was used. 7

Quantitative real-time RT-PCR analysis employed primers specific for Avr1k and the P. 8

sojae actin gene. Relative expression represents the ratio of Avr1k transcript levels 9

compared with that in mycelia, standardized to actin transcript levels. Bars represent 10

standard errors from two independent biological replicates each. 11

Fig. 6. Overexpression of Avr1k in P. sojae results in an avirulence phenotype on 12

soybeans containing Rps1k. 13

A, Verification of the transgene integration in transformant T-12 by amplification of 14

genomic DNA with oligonucleotides from the 5’ end of Avr1k and from the pHam34 15

terminator. WT = wild type recipient P7076; + = DNA template from the transformation 16

plasmid; - = no DNA template; M = size markers. B, Avr1k transcripts in mycelia of T-12 17

grown in vitro compared with P7076, detected by RT-PCR analysis. Amplification of 18

actin transcripts was used as a positive control. + = positive control consisting of plasmid 19

DNA template; - = no DNA template. C, Western blot assay of Avr1k protein in mycelia 20

of T-12 grown in vitro. Anti-His-tag monoclonal antibody was used to detect His-tagged 21

Avr1k protein in T12. In P7076, Avr1k is not His-tagged. Total protein was extracted 22

from in vitro-grown mycelia. Ponceau S staining verifies loading levels. D, Phenotypes in 23

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hypocotyl inoculation assays of P7076 and T-12 on soybean cultivars Williams (rps), and 1

Williams 82 (Rps1k, Williams background). Photographs were taken 4 days after 2

inoculation. Quantitative analysis of seedling killing is presented in Table 1. 3

Fig. 7 Gene silencing of Avr1k produces a virulence phenotype on soybeans containing 4

Rps1k. 5

A, Verification of the transgene integration in silencing transformants by amplification of 6

genomic DNA with oligonucleotides from the 5’ end of Avr1k and from the pHam34 7

terminator. WT = wild type recipient P6497; + = DNA template from transformation 8

plasmid; - = no DNA template; M = size markers. B, Avr1k transcript levels in 9

transformants compared with P6497, measured by quantitative real-time RT-PCR 10

analysis. Total RNA was extracted from infected soybean leaves at 12 hours post-11

inoculation. Levels of actin transcripts were used as a reference. Transcript levels relative 12

to actin were normalized to that of P6497 using the 7500 System Sequence Detection 13

Software. PCR reactions were replicated three times. Bars indicate standard errors. C, 14

phenotypes in representative hypocotyl inoculation assays of P6497 and transformants on 15

soybean cultivars Williams (rps), and Williams 82 (Rps1k, Williams background). 16

Photographs were taken 4 days after inoculation. T30 and T131 produced similar results 17

to T51 while the non-silenced transformant T32 produced similar results to P6497. 18

Quantitative analysis of seedling killing by all lines is presented in Table 1. 19

20

Supplementary Table S1. Functions of genes in the genomic region encompassing the 21

Avr1b/Avr1k locus 22

Supplementary Table S2. Oligonucleotides used in the study 23

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Supplementary Figure S1. Characterization of the P. sojae Avr1b/Avr1k locus 1

2

Supplementary Figure S2. Constructs for silencing of Avr1b. 3

Supplementary Figure S3. DNA sequence alignment of P. sojae Avr1k alleles 4

5

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Fig. 1. Identification of the RxLR effector gene Avr1k 165x115mm (300 x 300 DPI)

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Fig. 2. Cell death responses triggered by Avr1b-1 and Avr1k in the presence of Rps1b and Rps1k 82x48mm (300 x 300 DPI)

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Fig. 3. Overexpression of Avr1b-1 in P. sojae produces an avirulence phenotype on soybean cultivars containing Rps1k or Rps1b. 165x63mm (300 x 300 DPI)

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Fig. 4. Gene silencing of Avr1b-1 produces a virulence phenotype on soybean lines containing Rps1b or Rps1k

165x88mm (300 x 300 DPI)

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Fig. 5. Avr1k transcript levels during infection measured by quantitative real-time RT-PCR 76x44mm (300 x 300 DPI)

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Fig. 6. Overexpression of Avr1k in P. sojae results in an avirulence phenotype on soybeans containing Rps1k 127x100mm (300 x 300 DPI)

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Fig. 7 Gene silencing of Avr1k produces a virulence phenotype on soybeans containing Rps1k 127x77mm (300 x 300 DPI)

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Supplementary Figure S1. Genomic region encompassing the Avr1b/Avr1k locus

Scaffold_6: 3070000-3200000 from P. sojae sequence v5.0, scaffold_14: 320000-360000 from P. ramorum sequence v1.0 and

Supercontig 1.15: 2150000-275000 from P. infestans are shown. Black arrows indicate genes with syntenic orthologs in other

Phytophthora species, and white arrows show genes with no orthologs. The Avr1k and Avr1b-1 genes, and predicted transposable

elements are marked in this region of P. sojae. The pale blue bar indicates the location of BAC 3E16 to which genes responsible for

the Avr1b and Avr1k phenotypes were genetically localized by Shan et al (2004). Further details regarding the genes in this region are

given in Supplementary Table S1.

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Supplementary Figure S2. Constructs used for silencing of Avr1b.

A, Full length sense Avr1b-1 (nucleotides 1 to 417) driven by the constitutive Ham34

promoter

B, Full length antisense Avr1b-1 driven by the constitutive Ham34 promoter

C, Hairpin Avr1b-1 construct driven by the constitutive Ham34 promoter

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Avr1k_Race1(P6954) ATGATGCAATGGAGCGCAATCCTCATCCGCACTTGTTTCAGTGGCAGTGGAGGCGAGGCT







Avr1k_Race30 ATGATGCAATGGAGCGCAATCCTCATCCGCACTTGTTTCAGTGGCAGTGGAGGCaAGGCT

Avr1k_Race1(P6954) CTCACTTGCGCcACCTCCGAGCAAcAGACACGACCTGAATTATGTTTCTTCTTCAGCGTG



Avr1k_Race6(P7063) CTCACTTGCGCAACCTtCGAGCAAAAGACACGACCTGAATTATGTTTCTTCTTCAGCGTG



Avr1k_Race25(P7081) CTCACTTGCGCcACCTCCGAGCAAAAGACACGACgTGAATTATGTTTCTTCTTCAGCGTG

Avr1k_Race30 CTCACTTGCGCAACCTtCGAGCAAAAGACACGACCTGAATTATGTTTCTTCTTCAGCGTG

Avr1k_Race1(P6954) CGTTCCTCCTGGCCATCG--------ACCATCTCCGATGGTGCCTGTTTGGCACTCGTAT



Avr1k_Race6(P7063) CGTTCCTCCTGGCCATCGTGCTACTTACCATCTCCGATGGTGCCTGTTTGGCACTCGTAT

Avr1k_Race7(P7064) CGTTCCTCCTGGCCATCGTGCTACTTACCATCTCCGATGGTGCCTGTTTGGCACTCGTAT

Avr1k_Race19(P7076) CGTTtCTCCTGGCCATCGTGCTACTTACCATCTCCGATGGTGCCTGTTTGGCACTCGTAT

Avr1k_Race25(P7081) CGTTtCTCCTGGCCATCGTGCTACTTACCATCTCCGATGGTGCCTGTTTGGCACTCGTAT

Avr1k_Race30 CGTTCCTCCTGGCCATCGTGCTACTTACCATCTCCGATGGTGCCTGTTTGGCACTCGTAT

Avr1k_Race1(P6954) CGGCCGAACAAGGCGCCACGGCCGGGCGCAATACCTTATCCCTGCGATCGATGATGGCTA





Avr1k_Race19(P7076) CGGCCGAACAAGGCGCCACGGCCtGGCGCAATACCTTATCCCTGCGATCGATGATGGCTA

Avr1k_Race25(P7081) CGGCCGAACAAGGCGCCACGGCCtGGCGCAATACCTTATCCCTGCGATCGATGATGGCTA

Avr1k_Race30 CGGCCGAACAAGGCGCCACGGCCGGGCGCAATACCTTATCCCTGCGATCGATGATGGCTA

Avr1k_Race1(P6954) CTGAAGACATGGCAACCTCGACAAGATCACTACGATCTCAAGCTACGAACGTGGACGACG


Avr1k_Race11(P736 CTGAAGACATGGCAACCTCGACAAGATCACTACGATCTCAAGCTACGAACGTGGACGACG





Avr1k_Race30 CTGAAGACATGGCAACCTCGACAAGATCACTACGATCTCAAGCTACGAACGTGGACGACG

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Avr1k_Race1(P6954) ATGCTAACGTTTCGATTGAAAACAGAGGGATGAACCCTTCAGTCCTGACCAAGCTCGGCG


Avr1k_Race11(P7360 ATGCTAACGTTTCGATTGAAAACAGAGGGATGAACCCTTCAGTCCTGACCAAGCTCGGCG





Avr1k_Race30 ATGCTAACGTTTCGATTGAAAACAGAGGGATGAACCCTTCAGTCCTGACCAAGCTCGGCG

Avr1k_Race1(P6954) AATTTGCTTCGACGTTGACGGCTGGCAATACGGCGAACAAGTTATGGCTGATGGCTGACG







Avr1k_Race30 AATTTGCTTCGACGTTGACGGCTGGCAATACGGCGAACAAGTTATGGCTGATGGCTGACG

Avr1k_Race1(P6954) TGGACCCAAAGTCCGCGTTCAAGTTGCTGGGTCTTGACATGCCAGGGGTCCGCTTTATCG







Avr1k_Race30 TGGACCCAAAGTCCGCGTTCAAGTTGCTGGGTCTTGACATGCCAGGGGTCCGCTTTATCG

Avr1k_Race1(P6954) ACAACCCGAAGATGTTACAGTGGCTCAAGTTCACAAAAGCGTATTTAGACATGAAAAAGT





Avr1k_Race19(P7076) ACAACCCGAAGATGTTACAGTGGtTCAAGTTCACAAAAGCGTATTTAGACATGAAAAAGT

Avr1k_Race25(P7081) ACAACCCGAAGATGTTACAGTGGtTCAAGTTCACAAAAGCGTATTTAGACATGAAAAAGT

Avr1k_Race30 ACAACCCGAAGATGTTACAGTGGCTCAAGTTCACAAAAGCGTATTTAGACATGAAAAAGT

Avr1k_Race1(P6954) CGGGGTTTGGCGAGACAAGCGCACATGCTCTTCTGTATGAAAAAATTGGAGGACCCGACT



Avr1k_Race6(P7063) CGGGGTTTGGCGAGACAAGCGCACATGCTCTTCTGTATGAcAAAATTGGAGGACCCGACT

Avr1k_Race7(P7064) CGGGGTTTGGCGAGACAAGCGCACATGCTCTTCTGTATGAcAAAATTGGAGGACCCGACT



Avr1k_Race30 CGGGGTTTGGCGAGACAAGCGCACATGCTCTTCTGTATGAcAAAATTGGAGGACCCGACT

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Avr1k_Race1(P6954) TGTCACTACTTCTCCTAAGCCTGAAGGACGCTCCGGATGCGAACAGCTTGGTTCAAAAAC



Avr1k_Race6(P7063) TGTCACTACTTCTCCTAAGCCTGAAGGACGCTCCGGATGCGAACAGCTTGGTTaAAAAAC




Avr1k_Race30 TGTCACTACTTCTCCTAAGCCTGAAGGACGCTCCGGATGCGAACAGCTTGGTTCAAAAAC

Avr1k_Race1(P6954) TGACGAATTCTCAGTTTGGTATGTGGCATGACGCTCGAATCGAACCAGAACAGCTCGCAC







Avr1k_Race30 TGACGAATTCTCAGTTTGGTATGTGGCATGACGCTCGAATCGAACCAGAACAGCTCGCAC

Avr1k_Race1(P6954) AAACGGTGTTCAAAATTcAAGACGTCAGGAAACTACCTAAAAACGACCCAAAACTTCAAG



Avr1k_Race6(P7063) AAACGGTGTTCAAAATTGAAGACGTCAGGAAACTACCTAAAAACGACCCAAAACTTCAAG

Avr1k_Race7(P7064) AAACGGTGTTCAAAATTGAAGACGTCAGGAAACTACCTAAAAACGACCCAAAACTTCAAG

Avr1k_Race19(P7076) AAACGGTGTTCAAAATTGAAGACGTCAGGAAACTACCTAAAAACGACCCAAAACgTCAAG

Avr1k_Race25(P7081) AAACGGTGTTCAAAATTGAAGACGTCAGGAAACTACCTAAAAACGACCCAAAACgTCAAG

Avr1k_Race30 AAACGGTGTTCAAAATTGAAGACGTCAGGAAACTACCTAAAAACGACCCAAAACTTCAAG

Avr1k_Race1(P6954) TTATCGATGACTACGCGAAGTATCACAGAAAGCACCGGAAGTTTCTGAACAGCATCATGA



Avr1k_Race6(P7063) TTATCGATGACTACGCGAAGTATCACAGAgAGCACCGGAAGTTTCTGAACAGCATCATGA

Avr1k_Race7(P7064) TTATCGATGACTACGCGAAGTATCACAGAgAGCACCGGAAGTTTCTGAACAGCATCATGA

Avr1k_Race19(P7076) TTATCGATGACTACGCGAAGTATCACAaAAAcCACCGGgAGTTTCTGAACAGCATCATGA

Avr1k_Race25(P7081) TTATCGATGACTACGCGAAGTATCACAaAAAcCACCGGgAGTTTCTGAACAGCATCATGA

Avr1k_Race30 TTATCGATGACTACGCGAAGTATCACAGAgAGCACCGGAAGTTTCTGAACAGCATCATGA

Avr1k_Race1(P6954) TTATCTGA







Avr1k_Race30 TTATCTGA

Supplementary Figure S3. DNA sequence

alignment of P. sojae Avr1k alleles. Monomorphic

positions are highlighted in cyan, polymorphic

positions are highlighted in gray and white, with

variant bases shown in lower case.

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No. Gene IDa Location

a Annotation

b

E-

Valueb

Signal

Peptided

1 Physo3_249448 S6:3071857-3072754 novel plant SNARE 13-like [Glycine max] 1 E-13 None

2 Physo3_304891 S6:3073330-3074532R ankyrin repeat domain-containing protein 52

[Arthroderma gypseum] 9 E-08

None

3 Physo3_249376 S6:3074907-3075248 unknown None

4 Physo3_356688 S6:3075615-3076041R

DNA-directed RNA polymerase, putative[Phytophthora

infestans] 1 E-99

None

5 Physo3_514819 S6:3076503-3077243 Rab11 family GTPase, putative[Phytophthora infestans] 3 E-153 None


7 Physo3_318503 S6:3093488-3094213R

Thioredoxin domain-containing protein 9 [Ascaris

suum] 4 E-34 None

8 Physo3_564012 S6:3094477-3095491R

similar to PDGF associated protein [Ectocarpus

siliculosus] 1 E-24

None

9 No ID S6:3095782-3097025 thioredoxin H-type [Phytophthora infestans T30-4] 7 E-155 None

10 No ID S6:3097912-3099233 thioredoxin H-type [Phytophthora infestans T30-4] 7 E-155 None

11 Physo3_288365 S6:3101113-3101730 none None

12 Physo3_513270 S6:3103623-3105643

Intraflagellar Transport Protein 52 putative [Albugo

laibachii Nc14] 7 E-07 None


14 Physo3_249406 S6:3117496-3118566 putative deacetylase [Corynebacterium diphtheriae] 6 E-01 None

15 Physo3_512252 S6:3135220-3137007

phosphoribosylaminoimidazole carboxylase

[Phytophthora infestans] 0.0

None

16 Physo3_513314 S6:3137106-3138851R transmembrane protein 161B [Mus musculus] 3 E-18 None

17 Physo3_249437 S6:3138947-3139939

exosome complex exonuclease RRP4 [Phytophthora

infestans] 0.0

None

18 Physo3_512679 S6:3140766-3141809

putative phosphatidylinositol-4-phosphate binding

protein [Phytophthora sojae] 0.0

None

19 Physo3_288366 S6:3142483-3143322R Avh331 (RxLR-dEER protein) Yes

20 Physo3_288367 S6:3146161-3146577 Avr1b-1 (RxLR-dEER protein) 0.0 Yes

21 Physo3_337246 S6:3148643-3149118R unknown None

22 Physo3_337251 S6:3173236-3174199R unknown None

23 Physo3_514354 S6:3174467-3176409R

GPI mannosyltransferase 4, putative [Phytophthora

infestans] 0.0

None


25 No ID S6:3179000-3179986R

iojap-like ribosome-associated protein [Bartonella

melophagi] 2 E-15

None

26 Physo3_288369 S6:3180775-3182790R mucin-like protein [Phytophthora infestans] 0.0 None

27 No ID S6:3183690-3188702R mucin-like protein [Phytophthora infestans] 0.0 None

Supplementary Table S1. Functions of genes in the genomic region encompassing the Avr1b/Avr1k locus

a GeneIDs and scaffold (S) coordinates were obtained from Phytophthora sojae v3.0 genome database at

www.jgi.doe.gov (also called genome assembly v5.0 at http://www.eumicrobedb.org/). R indicates reverse

complement. b Annotation was performed using BlastX searches against the non-redundant protein database of NCBI, the

best annotated hits were taken as predicted function for the genes. c Signal peptide was predicted in SignalP 3.0 service (http://www.cbs.dtu.dk/services/SignalP-3.0/).

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No

.

Name Sequences (from 5' to 3’) a Usage

1 actinAF2 ACTGCACCTTCCAGACCATC Internal primers of P. sojae

Actin gene, for RT-PCR and real

time PCR mRNA assays

2 actinAR2 CCACCACCTTGATCTTCATG

3 UbI promoter-

Hind III -F

acaagcttGTTCCGTCATTTCCTCGCAG For the replacement of the

pHam34 promoter

4 UbI promoter-

Sma I -R

ttcccgggTGGATGCTCAGATGCTAGC

GTC

5 UbI promoter-F TCTGAGCCTCGCAGCTATTG Primers in Ubiquitin promoter

and pHam34 terminator, for

screening P. sojae transformants

6 pHam34-R AGACACAAAATCTGCAACTTC

7 Avr1k-RT-F CCACCTCCGAGCAACAGAC Internal primers of Avr1k, used

for real time PCR assay for

Avr1k mRNA

8 Avr1k-RT-R CCGATACGAGTGCCAAACA

9 Avr1k- His-F gggacaacaATGATGCAATGGAGCGC

AATCC

For insertion of Avr1k gene into

P. sojae transformation vector

10 Avr1K-His -R cggggtaccTCAGTGATGGTGATGGTG

ATGGATAATCATGATGCTGTTCAG

11 Avr1k-harf-F CGCAATCCTCATCCGCACTT Internal primers of Avr1k, used

for

RT-PCR assay for Avr1k mRNA

12 Avr1k-harf-R CGCTTGTCTCGCCAAACCC

13 Avr1bF ggggtaccgacaacaATGCGTCTATCTTTT

GTGCT

For insertion of anti-sense Avr1b

gene with P.sojae transformation

vector

14 Avr1bR ggggtaccTCAGCTCTGATACCGGTGA

A

15 Avr1bHPF: cacccgggtacCTTTTGTGCTTTCTCTTG For the amplification of hairpin

dsRNA of Avr1b 16 Avr1bHPR tgtctagaggATCCGTTGTAGATACGGT

CG

17 UblIF ttggatccTCGTTCGGGGTAAGTGGCG

18 UblIR tgtctagaTGCTAGCGTCACCGAACG

Supplementary Table S2. Summary of oligonucleotides used

a lower case bases indicate mismatches with the target sequences

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two rxlr avirulence genes in phytophthora sojae determine soybean ...

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