In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 108, No. 48 ( 2011-11-29)
Abstract:
Virulence proteins that reach beyond the pathogen–host interface to affect a third organism in the biological interaction have not yet been identified and functionally characterized. We found that SAP11 exerts a phenotype beyond the organism in which it resides, affecting not only the AY-WB–infected plant host but the reproductive success of the leafhopper vector that transmits AY-WB to a diverse range of plant species. Hence, SAP11 is a vivid example of the extended phenotype of the gene, a concept put forward in Richard Dawkins’ classic book ( 5 ). Nymph (or larva) production rates on the SAP11 transgenic and CIN-TCP –silenced Arabidopsis plants increased by 30–40%. Because leafhoppers produce 61% more nymphs on AY-WB–infected plants, additional virulence proteins of AY-WB also likely influence interactions between the leafhopper and the plant. Nonetheless, we consistently found an increase in oviposition rates and nymph production on all experimental plants, indicating that the SAP11-mediated destabilization of TCPs and impaired synthesis of JA promote leafhopper oviposition activity. Altogether, these results indicate that SAP11-mediated destabilization of CIN-TCPs leads to the down-regulation of LOX2 expression and JA synthesis and an increase in leafhopper egg-laying activity ( Fig. P1 ). We propose that SAP11 is a virulence protein that can enhance the likelihood of phytoplasma dispersal to other plants. Indeed, nymphs will hatch ∼10 d following egg laying on AY-WB–infected plants. These nymphs will feed immediately and will likely acquire the phytoplasmas. Systemic AY-WB infection of the leafhoppers is achieved when the insects are fairly developed nymphs or adults, all of which are capable of migrating to uninfected plant hosts and transmitting the phytoplasma. Therefore, phytoplasma strains producing virulence proteins, such as SAP11, will disperse faster and are likely to have a fitness advantage compared with phytoplasma strains without such proteins. Next, we examined how SAP11 affects plant defense against the leafhopper. CIN-TCPs positively regulate plant senescence, which is partly achieved through the up-regulation of a gene, LIPOXYGENASE2 ( LOX2 ), by TCP4 binding to LOX2 promoter sites ( 4 ). LOX2 encodes an enzyme, lipoxygenase, that mediates the first step of jasmonic acid (JA) synthesis. JA is an important plant hormone that has been reported to be involved in plant defense against various insects. Moreover, the expression of LOX2 is induced by infestation of the leafhopper and wounding. Because SAP11 negatively regulates CIN-TCPs, we examined the influence of SAP11 on LOX2 expression and JA accumulation. We determined that the LOX2 expression levels and the production of JA were considerably lower in the SAP11 lines than in WT Arabidopsis . Therefore, the presence of SAP11 is associated with the down-regulation of LOX2 and JA production. Furthermore, we observed that young leaves of AY-WB–infected Arabidopsis accumulate less JA compared with healthy Arabidopsis plants on wounding, which is consistent with the detection of SAP11 in young leaves of AY-WB–infected plants ( 1 ). Because we observed that leafhoppers produce more progeny on AY-WB–infected plants, we determined whether M. quadrilineatus shows increased nymph production on the SAP11 -expressing lines, on CIN-TCP – and LOX2 -silenced plants, and on Arabidopsis mutants that are impaired in the production of active JA. M. quadrilineatus produced more progeny on all the transgenic lines than on WT Arabidopsis . The increase in the number of progeny was attributable to the increased oviposition (egg-laying) activity of the female leafhoppers, as was also observed on AY-WB–infected plants. To find genetic evidence for TCP destabilization by SAP11, we obtained existing Arabidopsis lines ( 3 ) in which the expression of multiple CIN-TCP s is silenced ( 3 ) and compared their phenotypes with that of SAP11 -expressing plants. As we expected, the SAP11 and the silenced CIN-TCP plants exhibited similar leaf crinkling phenotypes. The crinkled leaf phenotypes of SAP11 plants were visibly reduced by overexpression of TCP4. These results support those of the biochemical analyses that SAP11 destabilizes CIN-TCPs. Next, we identified the target(s) of SAP11 in a plant host. We found that SAP11 predominantly interacted with two TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS 1 and 2 (TCP) transcription factors: TCP2 and TCP13. Similar TCP transcription factors are found among a wide range of plant species, and these regulate various aspects of plant development ( 2 ). Arabidopsis class I TCP members promote cell proliferation in leaves, whereas eight members of class II TCP [ CINCINNATA ( CIN ) -TCP s] negatively regulate cell proliferation but promote leaf maturation and aging (senescence). The balance between the two classes of TCPs determines plant morphology ( 3 ). TCP2 and TCP13 are both class II CIN-TCPs, indicating that SAP11 may target members within this class of TCPs. We predicted that SAP11 may target multiple TCPs via interaction with a protein structure (or domain) conserved in both classes of TCPs. This was confirmed by experiments demonstrating that SAP11 interacts with two class II CIN-TCPs (TCP2 and TCP4) and one class I TCP (TCP7). To investigate how SAP11 affects the TCPs, we used an assay system to express both SAP11 and TCPs in plant cells simultaneously in a transient manner. All eight class II CIN-TCPs were absent or decreased in abundance in the presence of SAP11 compared with the controls, whereas the abundance of class I TCP7 was similar between the SAP11 and control treatments. Altogether, these analyses indicate that the interaction of SAP11 with class II CIN-TCPs leads to destabilization of the latter, whereas the stability of class I TCP7 is not affected. To investigate the function of SAP11 further, we generated three genetically modified or transgenic Arabidopsis lineages (lines) that express SAP11 . These lines produced crinkled leaves and seed capsules and a greater number of stems compared with WT (naturally occurring) plants. This increase in stem production of the transgenic SAP11 lines is similar to the symptoms of witches’ broom observed in AY-WB–infected plants. Moreover, the severity of the changes in the observable features (phenotype) in each of the three transgenic SAP11 lines increased with increasing SAP11 expression levels (i.e., the line with the lowest SAP11 protein concentrations had the least severe phenotype). We used the model plant species Arabidopsis thaliana to investigate the mechanisms of phytoplasma virulence. Arabidopsis plants infected with AY-WB, compared with noninfected Arabidopsis plants, display a range of disease symptoms, including an increase in the number of stems (witches’ broom phenotype) and a 61% increase in the progeny numbers of the leafhopper vector. This increase in progeny is attributable to more eggs being laid by the adult female leafhoppers. Previously, we hypothesized that AY-WB phytoplasma secretes effector proteins that modulate specific targets (e.g., genes, proteins) in a host plant to alter its biology and influence interactions between the plant and the insect vector. An in-depth survey of the sequenced AY-WB genome, the sum of the genetic material of an organism, for genes encoding proteins secreted by the bacteria resulted in the identification of 56 candidate effector proteins, namely, SAPs ( 1 ). SAP11 is one of these 56 effector proteins. SAP11 was found to possess a protein signature that appeared to be important for the secretion of SAP11 from the bacteria and into the plant host. SAP11 also contains a protein signature important for targeting SAP11 to the plant nucleus during infection ( 1 ). Phytoplasmas are bacterial plant pathogens that are transmitted from plant to plant by specific insects (called vectors) that feed on nutrient-rich sap in the plant vascular tissue ( Fig. P1 ). Plants infected with phytoplasmas can display dramatic phenotypes or features that appear to benefit the insect vectors, such as the enhanced production of plant tissues that offer feeding and reproduction sources to the insect vectors. Because phytoplasmas depend on these insects for transmission to other plants, an increase in insect vector numbers would be beneficial to these pathogenic bacteria. Various examples are known of pathogens that alter the features of their primary animal or plant hosts to attract potential vectors, such as insects. However, the molecular mechanisms by which pathogens attract these vectors have not yet been elucidated. Plants infected with Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) are more susceptible than uninfected plants to the AY-WB vector leafhopper, Macrosteles quadrilineatus , which is a small plant-feeding insect; this insect produces more progeny (or offspring) on AY-WB–infected plants than on healthy ones. We report that the secreted AY-WB protein 11 (SAP11, a virulence protein or effector that aids the pathogen with its infection process and reproduction) produced by the bacteria, is at least partly responsible for this increase in the number of the insect vector progeny. The mechanism involves SAP11-mediated destabilization or reduced presence of the common transcription factors found among various plant species. These transcription factors are positive regulators of the defense response of the plant to leafhopper attack. Transcription factors are proteins that bind to specific DNA sequences within the promoter of a gene and regulate its expression.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1105664108
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2011
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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