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In nature, plants face enormous challenges from microbial pathogens and insect pests. Among them, phloem-feeding insects, such as aphids, cause serious damage to agriculture by depleting plant nutrients, modifying plant metabolism and vectoring pathogenic viruses (Blackman & Eastop, 2000). Aphid infestation usually causes limited damage to the plant in comparison with that caused by microbial pathogens and chewing insects. Aphids use their stylets to probe intercellularly through epidermal and mesophyll cell layers until they reach the phloem sieve element, where they establish a sustained interaction on suitable hosts (Tjallingii & Esch, 1993). Aphids probably use their specialized feeding tactics to minimize plant defence responses during the infestation process (reviewed by Thompson & Goggin, 2006; Goggin, 2007; Walling, 2008). Moreover, aphids may be able to manipulate plant physiology and, in particular, defence responses through the secretion of saliva into the phloem sieve elements to maintain their successful feeding on the plant (Will et al., 2007). Plant defence against aphids is complex, including premature leaf chlorosis and cell death (Pegadaraju et al., 2005). Such unique interactions represent an intriguing area in the study of plant interaction with other organisms. Understanding the nature of the plant’s ability to resist aphid feeding is of importance to agriculture and to our understanding of plant defence against biotic stresses.
Studies using the model plant Arabidopsis have contributed greatly to our understanding of resistance (R) gene-mediated plant defence, especially against pathogens (Jones & Dangl, 2006), as well as the basal defence mechanisms against aphid feeding (Pegadaraju et al., 2005, 2007; Couldridge et al., 2007; Kusnierczyk et al., 2008). With the identification and characterization of a large number of R genes and their corresponding pathogen effectors, a picture is emerging about the interactions of the two partners, as well as the downstream signalling cascades leading to full disease resistance (Takken et al., 2006; Schwessinger & Zipfel, 2008). Transcription factors (TFs) have been shown or implicated to be key regulators of the plant’s defence response (Hammond-Kosack & Parker, 2003; Gutterson & Reuber, 2004; McGrath et al., 2005; Eulgem & Somssich, 2007; Libault et al., 2007; Onate-Sanchez et al., 2007). Some members of specific TF families have been shown to be key components in the regulation of R gene-mediated disease resistance. For instance, Arabidopsis AtWRKY70 modulates the cross-talk between signalling pathways activating salicylic acid (SA)-dependent and repressing jasmonic acid (JA)-dependent responses, and is an essential element for both basal and full R gene-mediated resistance against the oomycete Hyaloperonospora parasitica (Li et al., 2004; J. Li et al., 2006; Knoth et al., 2007). TFs have also been implicated recently in the regulation of plant response to herbivory. Loss of function of Nicotiana attenuate WRKY3 or WRKY6 promotes the susceptibility of the plant to the chewing insect, Manduca sexta (Skibbe et al., 2008). In addition, overexpression of a rice TF gene, WRKY89, increases plant resistance to the white-backed planthopper Sogatella furcifera, a sap-sucking insect (Wang et al., 2007). Several cDNA or oligo-microarray experiments have shown that TFs are responsive to aphid feeding (De Vos et al., 2005; Levy et al., 2005; van Oosten, 2007; Kusnierczyk et al., 2008; Li et al., 2008). However, the limited sensitivity of these technologies has been a disadvantage in the accurate measurement of the low transcript level typical of TF genes. High-throughput quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains a sensitive and efficient method available for the quantification of large numbers of low-abundance TF genes. Such platforms have been established for the model plants Arabidopsis, rice and Medicago truncatula and have shown to be significantly more sensitive than microarrays (Czechowski et al., 2004; Caldana et al., 2007; Kakar et al., 2008).
The legume M. truncatula has emerged in recent years as an effective model for the study of R gene-mediated aphid resistance (Edwards & Singh, 2006). For example, a single M. truncatula line, Jester, has been shown to contain three independent R genes that confer strong resistance to three different aphid species (Klingler et al., 2005; Edwards & Singh, 2006; Gao et al., 2007a,b, 2008; Klingler et al., 2007; Guo et al., 2009; L. G. Kamphius, S. Guo & K. B. Singh, unpublished). Moreover, another M. truncatula line, A17, has been shown to contain additional R genes to bluegreen aphid (BGA; Acyrthosiphon kondoi) and pea aphid (PA; Acyrthosiphon pisum) (Klingler et al., 2009; Stewart et al., 2009). The aim of the present study was to provide an overview of the TF genes expressed during susceptible and resistant aphid interactions in M. truncatula, with a focus on the identification of TFs specifically involved in the early stages of aphid resistance. The strategy employed involved large-scale profiling of TF gene expression in closely related resistant and susceptible M. truncatula lines after BGA infestation, using qRT-PCR. The expression profiles of 752 putative TF transcripts were analysed at two early time points of the plant–aphid interaction. Comparison of the TF profiles between susceptible and resistant aphid interactions allowed us to identify large changes in TF gene expression associated with resistance following aphid infestation. Members of AP2/EREBP (pathogenesis-related transcriptional factor and ethylene response factor), bHLH (basic helix–loop–helix dimerization), C2H2 (Zn) (Zn-finger, C2H2 type) and WRKY (DNA-binding WRKY) gene families were specifically induced in the resistant plants after BGA infestation. Two genes from the bHLH and WRKY gene families were further analysed and shown to be associated with BGA resistance in other M. truncatula genetic backgrounds and with resistance to PA, suggesting that these TFs may play important roles in aphid resistance.
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This article provides an overview of the TF genes expressed during the early stages of BGA infestation in M. truncatula susceptible and resistant plants. Eighty TF genes belonging to 30 TF families were responsive to BGA infestation (Table 1). This large number of TF genes may reflect the complexity of aphid defence regulation and a broad spectrum of transcriptional reprogramming in the plants during the early stages of aphid infestation. Some of these TFs may be key regulators of aphid defence, and these may have gone undetected if relatively low-sensitive microarray techniques had been used in this study. Indeed, several microarray studies, mostly involving compatible plant–aphid interactions, have failed to identify or have identified a much smaller number of TFs in response to aphid infestation, although some of these were taken at later time points than in our study (Moran et al., 2002; Levy et al., 2005; Park et al., 2006; Couldridge et al., 2007; van Oosten, 2007; Kusnierczyk et al., 2008; Li et al., 2008).
TF profiling revealed different patterns of TF gene expression in M. truncatula during susceptible and resistant interactions with BGA (Table 1). The lack of transcriptional changes of TFs during the initial infestation (at 6 h) and the subsequent suppression of TFs during aphid feeding (at 12 h) in susceptible A17 plants provide further evidence at the molecular level that aphids may be able to avoid or suppress plant responses during the early infestation process in susceptible interactions (Walling, 2008). By contrast, there were a significantly larger number of TF genes differentially expressed in resistant Jester plants than in susceptible A17 plants on BGA infestation (61 genes in Jester vs 19 in A17). A significantly larger number of genes were also seen to be differentially expressed in resistant than susceptible soybean cultivars (not closely related) when the transcription profiling of 18 000 soybean genes was analysed at the same time points (at 6 and 12 h) on soybean aphid infestation (Li et al., 2008). This trend was also observed in incompatible and compatible interactions of soybean with avirulent and virulent strains of Pseudomonas syringae and other plant–pathogen systems (Zou et al., 2005; Klink et al., 2007). This suggests that resistant plants are able to trigger rapid and massive transcriptional reprogramming in order to hamper further infestation by aphids, which may be similar to plant defence against microbial pathogens. In the M. truncatula–BGA interaction, the responses of TF genes in resistant plants were as early as 6 h and were before the induction of defence-related genes, which occurred later than 12 h following aphid infestation (Gao et al., 2007a). This suggests that these TFs are among the early aphid-responsive genes in resistant plants. Some of the upregulated TFs are likely to represent key regulators that mediate the network of gene expression required for successful aphid resistance.
Among the suppressed TF genes in the BGA susceptible interaction, members of the HD-like and MYB/HD-like families showed relatively high representation (7/15). Downregulation of a MYB gene was also seen in tobacco (Nicotiana attenuata) after infestation by a chewing insect Manduca sexta (Hui et al., 2003). MYB/HD-like TF genes have been shown to have highly diversified biological functions (Jin & Martin, 1999; Yanhui et al., 2006), although there is relatively little information available with regard to their function in plant defence. In Arabidopsis, MYB family members have been shown to be responsive to plant defence elicitors and bacterial/fungal pathogens, whereas overexpression of a MYB gene increased plant resistance to a chewing insect, the fall armyworm (Spodoptera frugiperda), and a knockout of AtMYB102 resulted in enhanced susceptibility to the white cabbage butterfly (Pieris rapae) (Johnson & Dowd, 2004; McGrath et al., 2005; De Vos et al., 2006; Libault et al., 2007). In tobacco, a MYB gene has been shown to play a part in defence against tobacco mosaic virus (Hui et al., 2003). Other studies have suggested that the MYB family is one of the major TF families that regulate genes related to photosynthesis and metabolites (Grotewold, 2005; Allan et al., 2008; Malitsky et al., 2008; Saibo et al., 2009). It remains to be determined whether the downregulation of the MYB/HD-like genes observed in BGA-infested susceptible A17 plants is part of the aphid’s manipulation of the plant’s defence response to establish a successful feeding site. Alternatively, the downregulation of TF genes may play a role in the reduction of the photosynthetic rate and the suppression of genes related to photosynthesis, as shown in microarray studies involving compatible plant–aphid interactions (Macedo et al., 2003; Voelckel et al., 2004; Zhu-Salzman et al., 2004; Qubbaj et al., 2005; Botha et al., 2006; Eulgem, 2006; Macedo et al., 2009).
Among the upregulated TF genes in the resistant interaction, four gene families, AP2/EREBP, bHLH, C2H2 (Zn) and WRKY, showed relatively high representation (16/32), and the members from these TF families were exclusively induced in the resistant aphid interaction. Although transcription profiling studies of TF genes involved in R gene-mediated resistance have not been reported, the overrepresentation of the bHLH family in the resistant M. truncatula–BGA interaction appears to be distinct from the transcription signatures of TFs associated with plant defence responses in other studies (McGrath et al., 2005; Libault et al., 2007; Naoumkina et al., 2008). Although the specific members from each TF family varied depending on the interaction, the overrepresentation of AP2/EREBP and WRKY families among the upregulated TF genes was commonly observed in Arabidopsis plants in response to a fungal elicitor (e.g. chitin), a fungal pathogen Alternaria brassicicola and the plant hormone MeJA, as well as in M. truncatula cell suspension in response to a yeast elicitor (McGrath et al., 2005; Libault et al., 2007; Naoumkina et al., 2008). The C2H2 (Zn) family was among the overrepresented TF families in Arabidopsis in response to chitin (Libault et al., 2007). Members of AP2/EREBP, WRKY and C2H2 (Zn) families have also been shown to regulate plant defence responses against other biotic and abiotic stresses (Singh et al., 2002; Gutterson & Reuber, 2004; Eulgem, 2005; Qu & Zhu, 2006; Eulgem & Somssich, 2007).
Further evidence for the involvement of the MtWRKY and MtbHLH gene families more generally in aphid resistance was obtained by looking at additional independent interactions. The higher and/or exclusive induction of these genes was consistently observed in two other BGA-resistant M. truncatula cvs Caliph and Mogul, although the timing and magnitude of the induction varied depending on the genetic background (Figs 3a,b, 4a,b). This suggests that the induction of these MtbHLH and MtWRKY genes is probably linked to the R gene-mediated resistance against BGA. Our results (Fig. 2) on the M. truncatula line A17AKR+ (contains AKR and only differs from the susceptible line A17 by c. 1%) and JesterAKR− (no longer contains AKR and only differs from the resistant line Jester by c. 1%) further support this hypothesis. Members of the WRKY and bHLH families have also been implicated in the R gene-mediated resistance in soybean against soybean aphid (Li et al., 2008). In Arabidopsis, AtWRKY70 is required for both basal defence and full R gene (RPP4)-mediated disease resistance against the oomycete pathogen, Hyaloperonospora parasitica (Knoth et al., 2007).
Earlier and/or higher induction of the four TF genes was also seen in response to infestation by a different aphid species, PA (Fig. 7). In Jester, resistance to PA is mediated by a different gene, APR, than that mediating resistance to BGA (Gao et al., 2008; Guo et al., 2009; L. G. Kamphius, S. Guo & K. B. Singh, unpublished). The combined results for BGA and PA support a general role for bHLH and WRKY TFs in the mediation of aphid resistance, at least in M. truncatula. It is interesting to note that PA infestation induces these TF genes but, unlike the BGA response in Jester, the interaction does not seem to induce JA signalling (Gao et al., 2008). This suggests that these MtWRKY and MtbHLH members may act near the top of the JA signalling cascade of R gene-mediated aphid resistance or, alternatively, may function independently of the JA pathway. Consistent with this hypothesis was the observation that three of the four TFs examined did not respond to JA treatment, and therefore may act upstream of the octadecanoid pathway previously associated with BGA resistance in Jester (Gao et al., 2007a).
The specific function of the MtWRKY and MtbHLH genes in aphid resistance is yet to be characterized. Currently, there is a lack of information on the function of bHLH genes in relation to plant defence, whereas the members of the WRKY gene family have been relatively well studied, mostly in Arabidopsis (Eulgem et al., 2000; Toledo-Ortiz et al., 2003; Eulgem, 2005, 2006; X. Li et al., 2006; Eulgem & Somssich, 2007). Based on sequence similarity, MtWRKY (WRKY-1) and MtWRKY (WRKY-2) fall into Arabidopsis WRKY groups III and IIc, respectively (Eulgem et al., 2000). Interestingly, the three soybean WRKY genes which were induced by soybean aphid in a resistant cultivar, but not in a susceptible cultivar, also fall into the Arabidopsis WRKY group IIc (Li et al., 2008). Rice WRKY89 is a member of AtWRKY group III and has been implicated in resistance to the phloem-feeding white-backed leafhopper (Wang et al., 2007). All of these are in different Arabidopsis WRKY groups than tobacco WRKY3 and WRKY6 (WRKY group I), the silencing of which leads to increased susceptibility of tobacco plants to the chewing insect Manduca sexta via the JA signalling pathway (Skibbe et al., 2008). Thus, our WRKY TF results are consistent with the hypothesis that sucking insect defence resembles more closely that of pathogens rather than chewing insects (Walling, 2008). The discovery of TF genes associated with aphid resistance is important because they are likely to orchestrate genome-wide changes in transcription that lead to full R gene-mediated aphid resistance. For this reason, they are important targets for optimizing plant protection to aphids in agriculture.