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Molecular Genetics of Plant Microbe Interaction |
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» Research » Plant Microbe Interactions |
Employing Agrobacterium inoculation as a delivery system, we are introducing expression constructs containing various portions of the viral replicase into tobacco. These constructs are then scored for their ability to elicit N-dependent HR at the infiltration site. Through use of this technique it was demonstrated that a construct containing a C-terminal 50 kDa portion of the replicase was sufficient to elicit HR while constructs containing more minimal 43 and 46 kDa portions failed to trigger this response
Immunodetection of protein fragments: As a counterpart to the assay above, protein extracts from inoculated plants are prepared and subjected to immunoblot analysis. Replicase fragments were constructed with C-terminal FLAG epitopes to facilitate immunodetection. Interestingly, a 50 kDa fragment is easily visualized on a Western blot while the expected 43 and 46 kDa fragments are not. This result suggests that the failure to elicit could be due to a lack or instability of expressed protein. To address the possibility of protein instability, GFP fusion constructs were created. In a number of plant systems it has been demonstrated that expression of proteins as GFP fusions can produce enhanced stability without functional disruption of the fused protein of interest.
Agrobacterium harboring GFP (a negative control), GFP:p43-FLAG, and GFP:p50-FLAG constructs were generated. Inoculation with GFP strain failed to elicit an HR. Conversely, inoculation with the GFP:p50-FLAG strain led to formation of necrotic HR lesions in the infiltrated tissue. These findings strongly suggest that the failure of p43 to elicit is a function of low (or non-existent) protein levels If the recognition of the elicitor is structure dependent, than perhaps the GFP stabilized p43, though still containing sequences/domains essential for elicitation, is presenting a disrupted or overly labile structure. Conceivably this could reduce the number or affinity of interactions leading to host recognition.
Updated: 05/18/04
Plants use both preformed and inducible defense strategies to battle rogue microbes and insects. Gene-for-gene resistance is an inducible defense where pathogen recognition results from the specific interaction between a plant resistance gene and a pathogen gene. The presence of both of these genes induces a battery of defense responses that function to halt pathogen replication and spread. Our work focuses on the gene-for-gene interaction between tobacco mosaic virus (TMV) and plants containing the disease-resistance gene N. TMV infection of N-containing tobacco and tomato plants induces rapid defense responses, including cell death at the infection site, that limit viral replication, spread and disease. Specifically, my work addresses two fundamental questions concerning this interaction: 1) How does TMV infection trigger the host defense responses? 2) What is the role of the N gene product in TMV recognition and defense?
Previous work from Roger Beachys group showed that the TMV replicase proteins are involved in triggering the N gene-mediated defense responses. We have confirmed and extended this finding by showing that transient, non-viral expression of a TMV replicase gene fragment containing the putative helicase domain is sufficient to elicit N-mediated cell death (Abbink et al., 1998, MPMI 11:12, 1242-1246; Erickson et al., 1999, Plant J. 18:1, 67-76.). This discovery was further verified using transgenic plants, where tobacco seedlings expressing this TMV replicase fragment (termed p50) exhibited N gene-dependent cell death throughout the plant, killing the seedlings within two weeks after germination. Like the defense responses elicited by TMV infection, p50 expression induces a temperature-sensitive cell death response.
- Abbink et al., 1998, MPMI 11:12, 1242-1246; Erickson et al., 1999, Plant J. 18:1, 67-76.
Updated: 05/18/04
Biochemical analyses in vitro analysis of recombinant p50 protein shows it can hydrolyze ATP, as predicted by the presence of ATPase/helicase motifs located in this portion of the replicase proteins. A point mutation in one of these motifs (the P-loop) abolishes ATPase activity, but does not alter cell death-inducing activity, indicating enzymatic activity is not required for elicitation. Taken together, these observations suggest that structural features of the helicase domain of the TMV replicase proteins are recognized during viral infection by N-containing plants, and this recognition elicits the defense responses.
The N gene is similar in nucleotide sequence to other gene-for-gene type of resistance genes (R genes) that confer resistance to a diverse group of pathogens including bacteria, fungi, viruses, insects and nematodes. Because of their specific role in pathogen defense and their predicted protein structures, R genes have been hypothesized to encode receptors that directly or indirectly recognize pathogen elicitor molecules and signal for defense responses. Work is ongoing using N and p50 to test these ideas.
Updated: 05/18/04
 | | N Mediated TMV Resistance is attenuated in NPKI- plants |
The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. The conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Mutation of the conserved basic amino acids in this motif, or the deletion of N-terminal 64 amino acids containing this motif significantly compromised or abolished the ability of NtMEK2DD to activate SIPK/WIPK in vivo. These mutants were also defective in interacting with SIPK and WIPK, suggesting protein-protein interaction is required for the functional integrity of this MAPK cascade. To eliminate Agrobacterium that is known to activate a number of defense responses in transient transformation experiments, we generated permanent transgenic plants. Induction of NtMEK2DD expression by dexamethasone induced HR-like death in both T1 and T2 plants. In addition, by using PVX-induced gene silencing, we demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. Together with previous reports that SIPK and WIPK are activated by TMV in a gene-for-gene-dependent manner, we conclude that NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death.
Mitogen activated protein kinase (MAPK) cascades are rapidly activated upon plant recognition of invading pathogens. Here, we describe the use of virus induced gene silencing (VIGS) to study the role of candidate plant MAP kinase kinase kinase (MAPKKK) homologs of human MEKK1 in pathogen-resistance pathways. We demonstrate that silencing expression of a tobacco MAPKKK, Nicotiana Protein Kinase 1 (NPK1), interferes with the function of the disease resistance genes N, Bs2 and Rx, but does not affect Pto- and Cf4-mediated resistance. Further, NPK1-silenced plants also exhibit reduced cell size, defective cytokinesis, and an overall dwarf phenotype. Out results provide evidence that NPK1 functions in the regulation of N-, Bs2-, and Rx-mediated resistance responses and may play a role in one or more MAPK cascades, regulating multiple cellular processes.
- Jin, H., Axtell, M. J., Dahlbeck, D., Ekwenna, O., Zhang, S., Staskawicz, B., Baker, B. (2002) NPK1, an MEKK1-like mitogen-activated protein kinase kinase kinase, regulates innate immunity and development in plants Dev. Cell 3:291-297
Updated: 05/18/04
The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. The conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Mutation of the conserved basic amino acids in this motif, or the deletion of N-terminal 64 amino acids containing this motif significantly compromised or abolished the ability of NtMEK2DD to activate SIPK/WIPK in vivo. These mutants were also defective in interacting with SIPK and WIPK, suggesting protein-protein interaction is required for the functional integrity of this MAPK cascade. To eliminate Agrobacterium that is known to activate a number of defense responses in transient transformation experiments, we generated permanent transgenic plants. Induction of NtMEK2DD expression by dexamethasone induced HR-like death in both T1 and T2 plants. In addition, by using PVX-induced gene silencing, we demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. Together with previous reports that SIPK and WIPK are activated by TMV in a gene-for-gene-dependent manner, we conclude that NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death.
 | | Suppression of NtMEK2, SIPK or WIPK expression via PVX virus-induced gene silencing compromised N gene mediated resistance. N. benthamiana::NN plants were infiltrated with Agrobacterium carrying binary PVX constructs with fragment of N (a positive control), NtMEK2, SIPK, WIPK or SIPKK (a negative control). Fourteen days later, the upper leaves that show the silencing of target gene were inoculated with GFP-tagged TMV. The presence/ spreading of the TMV virus in both the inoculated leaf and the upper inoculated leaf was determined 5 days later by visualizing the green fluoresces under UV light. |
- Jin, H., Liu, Y., Yang, K., Kim, C. Y., Baker, B., Zhang, S. (2003) Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco The Plant Journal 33:719-731
Updated: 05/18/04
 | | Depiction of the functions and interrelationship between SIPK and WIPK in tobacco defense signaling. |
Plant mitogen-activated protein kinases (MAPKs) represented by tobacco wounding-induced protein kinase (WIPK) have unique regulation at the level of transcription in response to stresses. By using transcriptional and translational inhibitors, it has been shown previously that WIPK gene expression and de novo protein synthesis are requires for the high-level activity of WIPK in cells treated with elicitins from Phytophthora spp. However, regulation of WIPK expression and the roles (s) of WIPK in plant disease resistance are unknown. We demonstrated that WIPK gene transcription is regulated by phosphorylation and de-phosphorylation events. Interestingly, salicylic acid-induced protein kinase (SIPK) was identified as the kinase involved in regulating WIPK gene expression based on both gain-of-function and loss-of-function analyses. This finding revealed an additional level of interaction between SIPK and WIPK, which share and upstream MAPKK, NtMEK2. Depending on whether WIPK shares its downstream targets with SIPK, it could either function as a positive feed-forward regulator of SIPK or initiate a new pathway. Consistent with the first scenario, co-expression of WIPK with the active mutant of NtMEK2 leads to accelerated hypersensitive response (HR)-like cell death in which SIPK also plays a role. Mutagenesis analysis revealed that the conserved common docking domain in WIPK is required for its function. Together with prior reports that (i) WIPK is activated in NN tobacco infected with tobacco mosaic virus, and (ii) PVX virus-induced gene silencing of WIPK attenuated N gene-mediated resistance, we concluded that WIPK plays a positive role in plant disease resistance, possibly through accelerating the pathogen-induced HR cell death.
- Liu, Y., Jin, H., Yang, K., Kim, C. Y., Baker, B. and S. Zhang (2003) Interaction between two mitogen-activated protein kinases during tobacco defense signaling The Plant Journal 2:149-160
Updated: 05/18/04
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» Research » Plant Microbe Interactions |
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