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. 2009 Aug 20;10(6):837–842. doi: 10.1111/j.1364-3703.2009.00577.x

Contributions of the effector gene hopQ1‐1 to differences in host range between Pseudomonas syringae pv. phaseolicola and P. syringae pv. tabaci

PATRIZIA FERRANTE 1, CHRISTOPHER R CLARKE 2, KERI A CAVANAUGH 3, RICHARD W MICHELMORE 3, ROBERTO BUONAURIO 1, BORIS A VINATZER 2,✉
PMCID: PMC6640246  PMID: 19849789

SUMMARY

To study the role of type III‐secreted effectors in the host adaptation of the tobacco (Nicotiana sp.) pathogen Pseudomonas syringae pv. tabaci, a selection of seven strains was first characterized by multilocus sequence typing (MLST) to determine their phylogenetic affinity. MLST revealed that all strains represented a tight phylogenetic group and that the most closely related strain with a completely sequenced genome was the bean (Phaseolus vulgaris) pathogen P. syringae pv. phaseolicola 1448A. Using primers designed to 21 P. syringae pv. phaseolicola 1448A effector genes, it was determined that P. syringae pv. phaseolicola 1448A shared at least 10 effectors with all tested P. syringae pv. tabaci strains. Six of the 11 effectors that failed to amplify from P. syringae pv. tabaci strains were individually expressed in one P. syringae pv. tabaci strain. Although five effectors had no effect on phenotype, growth in planta and disease severity of the transgenic P. syringae pv. tabaci expressing hopQ1‐1 Pph1448A were significantly increased in bean, but reduced in tobacco. We conclude that hopQ1‐1 has been retained in P. syringae pv. phaseolicola 1448A, as this effector suppresses immunity in bean, whereas hopQ1‐1 is missing from P. syringae pv. tabaci strains because it triggers defences in Nicotiana spp. This provides evidence that fine‐tuning effector repertoires during host adaptation lead to a concomitant reduction in virulence in non‐host species.

Many plant pathogens that live extracellularly between plant cells translocate highly specialized proteins, called effectors, into plant cells, where they suppress the plant immune system as part of their pathogenesis (Gohre and Robatzek, 2008). The plant pathogen Pseudomonas syringae and many other phytobacteria use a type III secretion system (T3SS) for the translocation of effectors into host cells (Cunnac et al., 2009). Bacteria unable to translocate effectors, either because they are naturally missing such ability or because they are mutated in their T3SS, are successfully inhibited in their growth by the plant immune system and do not cause disease (for example, see Mohr et al., 2008).

The plant defence system is currently considered to consist of two types of immune response. The first response is triggered by conserved microbial‐associated molecular patterns (MAMPs), with the best‐known example being flagellin (Zipfel et al., 2004). This immune response is called MAMP‐triggered immunity (MTI) (Chisholm et al., 2006). The second response is triggered by some of the effectors translocated into plant cells ‘intended’ by the pathogens to suppress MTI. This response is designated ETI (effector‐triggered immunity) (Chisholm et al., 2006). Although MTI appears to be a general response triggered by many microbes on most plants, ETI only occurs when plants express a cognate resistance (R) protein that can directly or indirectly detect the presence of a specific pathogen effector. ETI is often, but not always, accompanied by a plant hypersensitive response (HR), which consists of programmed cell death accompanied by a range of cellular and biochemical changes that limit pathogen growth (Greenberg, 1997). The HR is macroscopically visible as leaf collapse when a large number of avirulent bacteria are infiltrated into a leaf. An additional layer of complexity in plant–pathogen interactions is added by some effectors abrogating ETI (Jamir et al., 2004).

As plant pathogenic bacteria are under selection pressure to evade immunity, and plants are under antagonistic selection pressure to become, or to remain, immune, bacteria have evolved diverse overlapping repertoires of effectors, and plants have evolved diverse complements of resistance proteins capable of recognizing subsets of effectors (Wroblewski et al., 2009). Individual strains of P. syringae express dozens of effectors (Cunnac et al., 2009; Guttman et al., 2002), and individual plants contain hundreds of resistance gene candidates (McHale et al., 2006). This co‐evolution of plants and pathogens is often considered as an arms race (Stavrinides et al., 2008).

Selection during evolution will fine tune the effector repertoire of each pathogen to maximize virulence on its hosts by retaining/acquiring a set of effectors to suppress immunity on its hosts and losing effectors that are recognized by resistance proteins of its hosts. An effector repertoire optimized for one plant species may include effectors that trigger immunity on other plant species (Wroblewski et al., 2009). Consequently, the adaptation of a pathogen to its hosts will tend to lead to reduced virulence on other plant species and a progressive narrowing of the host range of each pathogen. Although this is an obvious scenario explaining the general basis of host specificity in plant pathogens, there are few data supporting this hypothesis. In a survey of the reactions of host and non‐host genotypes to effector repertoires from multiple bacterial pathogens, non‐host species were shown to react to multiple effector proteins from individual pathovars more frequently and more intensely than host species (Wroblewski et al., 2009); however, complementary data from pathogens were lacking. Moreover, although several effectors with avirulence activity on some plants have been shown to suppress immunity on other plants [for example, AvrRpt2 (Guttman and Greenberg, 2001; Lim and Kunkel, 2005), AvrPto1 (Chang et al., 2000) or AvrPphF (Tsiamis et al., 2000)], the absence or presence of such effectors in closely related pathogens that infect plant species on which an effector triggers or suppresses immunity, respectively, has not been analysed. For example, the tomato pathogen DC3000 expresses the effector hopQ1‐1, which triggers immunity in the non‐host Nicotiana benthamiana (Wei et al., 2007). However, it is not known whether this effector suppresses immunity in the host tomato, or whether hopQ1‐1 is missing from strains closely related to DC3000 that cause disease on plants in which HopQ1‐1 triggers immunity.

In this article, we report the identification of differences in the effector repertoires between the bean pathogen P. syringae pv. phaseolicola 1448A (Pph1448A) and the tobacco pathogen P. syringae pv. tabaci (Pta) DAPP‐PG677. We show how the effector hopQ1‐1 absent in all analysed Pta strains, but present in Pph1448A, triggers immunity in tobacco, but increases virulence in bean, consistent with the fine tuning of pathogen effector repertoires to the defence system of their hosts.

First, multilocus sequence typing (Maiden et al., 1998) was used to type five Pta strains isolated from cultivated tobacco (N. tabacum) in Italy in 2005 (PtaDAPP‐PG 673–677), one strain isolated in Hungary in 1959 (the pathovar tabaci pathotype strain LMG 5393) and one strain isolated in Malawi in 1971 (Pta LMG5192). Detailed materials and methods are published as Supporting Information. All strains had identical sequences at the rpoD, gyrB, acnB and pgi loci used by Yan et al. (2008). Moreover, they were identical to the Pta strain MAFF301612 collected in Japan in 1967 analysed by Hwang et al. (2005). Pathovar tabaci thus appears to be a monophyletic subgroup of P. syringae of recent evolutionary origin. However, as it is difficult to estimate how many generations a year a plant pathogen undergoes (Wichmann et al., 2005), and as we did not find any nucleotide differences between strains to calculate genetic distances between strains, it is impossible to estimate how many years have passed since the most recent common ancestor of today's Pta strains. Only the identification of single nucleotide polymorphisms on a genome‐wide scale between geographically separated Pta strains will make it possible to calculate such a time estimate, as was recently performed for the human and animal pathogen, Bacillus anthracis (Van Ert et al., 2007).

Based on the gene fragments used by Hwang et al. (2005), the analysed Pta strains are closely related to the completely sequenced bean pathogen Pph1448A (Joardar et al., 2005), with over 98% DNA identity over the entire length of the four sequenced gene fragments. Figure 1 shows the branch of a neighbour‐joining tree containing Pph1448A and all the Pta isolates. The full P. syringae tree built with the four concatenated gene fragments of all strains used by Hwang et al. (2005), downloaded from the Plant‐Associated Microbes database (PAMDB) website (http://www.pamdb.org), is shown in Fig. S1 (see Supporting Information). The Pta isolates were much more distantly related to the other two completely sequenced P. syringae strains PtoDC3000 (Buell et al., 2003) and P. syringae pv. syringae B728a (Feil et al., 2005), with percentage DNA identity values of approximately 92% and 94%, respectively.

Figure 1.

Figure 1

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Clade of a phylogenetic tree of Pseudomonas syringae constructed with the concatenated dataset of gene fragments of the four housekeeping genes gap1, gltA, gyrB and rpoD (Hwang et al., 2005). The clade contains Pph1448A (‘phaseolicola 1448’) and the analysed strains of pathovar tabaci (pathotype strain LMG5393 is shown as a representative).

As the T3SS effector repertoire of Pph1448A had been well characterized (Vencato et al., 2006), we were able to design primers for 21 of these effectors (Table S1, see Supporting Information), and determined, by polymerase chain reaction (PCR), the presence of 10 of them in all analysed Pta strains: hopR1, hopAE1, hopAH2, hopAN1, hopAS1, hopAJ2, hopD1, hopI1, hopX1 and hrpK1 (data not shown). However, we did not obtain PCR products from any Pta strain with primers that were designed for the effectors hopAB1, hopAF1, hopAJ1, hopG1, hopF3, hopQ1‐1, avrRps4, avrD1, avrB2, avrB4‐1 and avrB4‐2. Therefore, DNA sequences of these effectors are either sufficiently different in Pta such that our primers did not anneal, or these effectors are absent from Pta. Because the nucleotide sequence identity between Pta strains and Pph1448A is over 98% for the four housekeeping genes (see previous paragraph), it is likely that many or all of the undetected effector‐encoding genes are missing from the analysed Pta strains. Their absence was confirmed by searching the recently released draft genome sequence of the Pta strain ATCC11528 (NZ_ACHU00000000); none of the effector genes undetected by PCR were found in this additional strain of Pta based on blast analysis (Altschul et al., 1997).

To determine whether the likely absence of the above effectors from Pta strains, and their known presence in Pph1448A, may contribute to the difference in host specificity of these strains, a subset of these effectors (avrB4‐1, avrB2, avrRps4, hopF3, hopAF1, and hopQ1‐1) was cloned from Pph1448A and expressed in a strain of Pta: DAPP‐PG677. Leaves of N. benthamiana and bean were inoculated with the six isogenic transgenic Pta strains, each expressing one of these effectors, with PtaDAPP‐PG677, or with Pph1448A. Pta strains expressing avrB4‐1, avrB2, avrRps4, hopF3, hopAF1 did not show significant differences, in either symptoms (data not shown) or their growth in planta, compared with PtaDAPP‐PG677 carrying the empty vector pME6010 (Fig. 2A,B). However, the inoculation of leaves of N. benthamiana and bean with the transgenic PtaDAPP‐PG677 expressing hopQ1‐1 resulted in less severe, and more severe, symptoms, respectively, compared with PtaDAPP‐PG677 carrying only the empty vector (Fig. 3A,B).

Figure 2.

Figure 2

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Growth of transgenic Pseudomonas syringae pv. tabaci (Pta) strains expressing effectors from P. syringae pv. phaseolicola 1448A (Pph1448A) compared with the progenitor PtaDAPP‐PG677 and Pph1448A on Nicotiana benthamiana (A) and bean (B), and the growth of PtaDAPP‐PG677 and PtaDAPP‐PG677 expressing HopQ1‐1 on N. tabacum (C). Each value is the mean of six (A, B) or 12 (C) replicates ± standard error. Columns within each plot capped with the same letters are not significantly different according to Duncan's multiple range test (A, B; P < 0.05) and F‐test (C; P < 0.01). It should be noted that the Pta strains expressing effectors need to be compared with Pta carrying the empty vector pME6010, as effectors were expressed from a vector based on pME6010, and pME6010 by itself reduces the growth of Pta.

Figure 3.

Figure 3

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Symptom differences caused by the expression of hopQ1‐1 in Pseudomonas syringae pv. tabaci DAPP‐PG677 (PtaDAPP‐PG677) on Nicotiana benthamiana (A) and bean (B) 72 h after low‐dose infection. The hypersensitive response (HR) test of PtaDAPP‐PG677 (labelled as Pta) and PtaDAPP‐PG677 expressing hopQ1‐1 (labelled as Pta+hopQ1‐1) on N. benthamiana (C) and N. tabacum (D) 18 and 24 h, respectively, after high‐dose infection. The circles in each photograph show where the syringe was pressed against the leaf to infiltrate bacteria. In (A), areas of chlorosis on the upper leaf surface (left) infected with Pta are indicated by arrows. No chlorosis is visible in the area infiltrated with Pta+hopQ1‐1. In addition, the lower leaf surface (right) infiltrated with Pta is rough, whereas the lower leaf surface of the area infiltrated with Pta+hopQ1‐1 is smooth. In (B), the entire upper leaf area (left) infiltrated with Pta+hopQ1‐1 exhibits homogeneous chlorosis, whereas Pta does not cause any chlorosis. On the lower side of the leaf (right), most of the leaf area infiltrated with Pta+hopQ1‐1 is rough, whereas the area infiltrated with Pta is significantly healthier. In (C), areas of water soaking can be seen extending over almost the entire area infiltrated with Pta, but not in the area infiltrated with Pta+hopQ1‐1. In (D), the tissue infiltrated with Pta+hopQ1‐1 has collapsed, whereas the tissue infiltrated with Pta has not.

Measurement of bacterial growth in planta confirmed the positive effect on virulence of hopQ1‐1 in bean and the negative effect in N. benthamiana. The growth of transgenic PtaDAPP‐PG 677 expressing hopQ1‐1 was significantly reduced in N. benthamiana leaves and significantly increased in bean leaves, when compared with the growth of PtaDAPP‐PG677 carrying the empty vector (Fig. 2A,B).

As mentioned above, the gene hopQ1‐1 of PtoDC3000 had been shown previously to reduce the growth of the Pta strain ATCC11528 and to induce an HR in N. benthamiana (Wei et al., 2007). Moreover, hopQ1‐1 Pph1448A, when transiently expressed using Agrobacterium tumefaciens, induces necrosis in N. tabacum and chlorosis in N. benthamiana (Wroblewski et al., 2009). Therefore, we determined whether PtaDAPP‐PG677 expressing hopQ1‐1 Pph1448A would induce an HR in N. benthamiana and N. tabacum following inoculation at high concentration. In agreement with the transient assays described by Wroblewski et al. (2009), no HR was detected in N. benthamiana; by contrast, PtaDAPP‐PG677 induced more severe and faster necrosis than PtaDAPP‐PG677 expressing hopQ1‐1 Pph1448A (Fig. 3C). As this observation correlates with the reduction in symptoms and bacterial growth caused by the expression of hopQ1‐1 when Pta is inoculated at a low dose, this necrosis should be interpreted as disease rather than an HR. On N. tabacum, PtaDAPP‐PG677 expressing hopQ1‐1 Pph1448A caused an HR, whereas PtaDAPP‐PG677 did not (Fig. 3D), which is again in agreement with the HR induced by hopQ1‐1 in the A. tumefaciens‐mediated transient assay. When following up on these results with low‐dose infections of N. tabacum, PtaDAPP‐PG677 expressing hopQ1‐1 Pph1448A grew significantly less than wild‐type PtaDAPP‐PG677 (Fig. 2C). However, the growth reduction was only twofold.

Our results are in agreement with the hypothesis that, as strains of P. syringae adapt to host species and maximize virulence on those species, they concomitantly compromise their ability to cause disease on other potential hosts, consequently reducing their host range. In the case of Pta, not expressing hopQ1‐1 is a selective advantage, as hopQ1‐1 reduces the growth of Pta on its host tobacco; however, not expressing hopQ1‐1 reduces the virulence on bean and possibly on other species on which HopQ1‐1 does not trigger a resistance response. Therefore, not expressing hopQ1‐1 reduces the fitness of Pta on these species. However, expressing hopQ1‐1 is a selective advantage for Pph1448A, as hopQ1‐1 contributes to virulence on bean. However, expressing hopQ1‐1 probably reduces the ability of Pph1448A to reproduce on tobacco and, possibly, on other plant species on which it triggers a resistance response, reducing its fitness on these species. Although a reduction in bacterial growth on a plant species because of the acquisition or loss of a single effector may not immediately exclude that plant species from the host range of a P. syringae strain, even small growth differences in controlled conditions may have conspicuous effects on fitness in the field (Wichmann and Bergelson, 2004). Therefore, sequential acquisitions and losses of effectors with quantitative effects on virulence can be expected to lead to significant increases in virulence on some plant species and a concomitant reduction in virulence on other plant species, narrowing the host range of a strain to a small number of species.

It would be interesting to delete hopQ1‐1 from Pph1448A to determine whether a role for hopQ1‐1 in the pathogenesis of Pph1448A on bean can be confirmed. However, a negative result may simply mean that other effectors in Pph1448A have redundant function. Redundancy between effectors may also be the reason why the Pta strains expressing Pph1448A effector genes other than hopQ1‐1 have no phenotype.

With a draft genome sequence of PtaATCC11528 now available, it will also be possible to compare the genomic regions containing effectors in PtaATCC11528 and Pph1448A in order to determine by which mechanisms effectors are acquired and/or lost by these two strains, for example through the acquisition or loss of genomic islands (Lindeberg et al., 2008; Pitman et al., 2005). Moreover, identifying the mechanism by which N. benthamiana triggers immunity on perception of HopQ1‐1 may lead to the development of new approaches to render bean more resistant to Pph1448A.

Supporting information

Appendix S1 Materials and methods.

Fig. S1. Phylogenetic tree based on data by Hwang et al. (2005) and downloaded from http://www.pamdb.org.

Table S1 Pph1448A effector genes for which the presence in strains of Pta was detected by polymerase chain reaction.

Please note: Wiley‐Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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ACKNOWLEDGEMENTS

This work was supported by a National Science Foundation CAREER award to B.A. Vinatzer (grant number 0746501), a National Science Foundation Plant Genome award to R.W. Michelmore and J. T. Greenberg (grant number 0211923) and a grant from British American Tobacco Italy to R. Buonaurio.

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Associated Data

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Supplementary Materials

Appendix S1 Materials and methods.

Fig. S1. Phylogenetic tree based on data by Hwang et al. (2005) and downloaded from http://www.pamdb.org.

Table S1 Pph1448A effector genes for which the presence in strains of Pta was detected by polymerase chain reaction.

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