Fumiaki Katagiri

A High-Throughput Arabidopsis Reverse Genetics System

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from ∼100,000 transformed lines. A total of 85,108 TAIL-PCR products from 52,964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.

The A. thaliana disease resistance gene RPS2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats

In plants, resistance to a pathogen is frequently correlated with a genetically defined interaction between a plant resistance gene and a corresponding pathogen avirulence gene. A simple model explains these gene-for-gene interactions: avirulence gene products generate signals (ligands), and resistance genes encode cognate receptors. The A. thaliana RPS2 gene confers resistance to the bacterial pathogen P. syringae carrying the avirulence gene avrRpt2. A map-based positional cloning strategy was used to identify RPS2. The identification of RPS2 was verified using a newly developed transient assay for RPS2 function and by genetic complementation in transgenic plants. RPS2 encodes a novel 105 kDa protein containing a leucine zipper, a nucleotide-binding site, and 14 imperfect leucine-rich repeats.

Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae

We performed large-scale mRNA expression profiling using an Affymetrix GeneChip to study Arabidopsis responses to the bacterial pathogen Pseudomonas syringae. The interactions were compatible (virulent bacteria) or incompatible (avirulent bacteria), including a nonhost interaction and interactions mediated by two different avirulence gene–resistance (R) gene combinations. Approximately 2000 of the ∼8000 genes monitored showed reproducible significant expression level changes in at least one of the interactions. Analysis of biological variation suggested that the system behavior of the plant response in an incompatible interaction was robust but that of a compatible interaction was not. A large part of the difference between incompatible and compatible interactions can be explained quantitatively. Despite high similarity between responses mediated by the R genes RPS2 and RPM1 in wild-type plants, RPS2-mediated responses were strongly suppressed by the ndr1 mutation and the NahG transgene, whereas RPM1-mediated responses were not. This finding is consistent with the resistance phenotypes of these plants. We propose a simple quantitative model with a saturating response curve that approximates the overall behavior of this plant-pathogen system.

The Arabidopsis thaliana-pseudomonas syringae interaction.

Pseudomonas syringae is a Gram-negative, rod-shaped bacterium with polar flagella (Figure 1; Agrios, 1997). Strains of P. syringae collectively infect a wide variety of plants. Different strains of P. syringae, however, are known for their diverse and host-specific interactions with plants (Hirano and Upper, 2000). A specific strain may be assigned to one of at least 40 pathovars based on its host range among different plant species (Gardan et al., 1999) and then further assigned to a race based on differential interactions among cultivars of the host plant. Understanding the molecular basis of this high level of host specificity has been a driving force in using P. syringae as a model for the study of host-pathogen interactions. In crop fields, infected seeds are often an important source of primary inoculum in P. syringae diseases, and epiphytic bacterial growth on leaf surfaces often precedes disease development (Hirano and Upper, 2000). P. syringae enters the host tissues (usually leaves) through wounds or natural openings such as stomata, and in a susceptible plant it multiplies to high population levels in intercellular spaces. Infected leaves show water-soaked patches, which eventually become necrotic. Depending on P. syringae strains, necrotic lesions may be surrounded by diffuse chlorosis. Some strains of P. syringae also cause cankers and galls (Agrios, 1997). In resistant plants, on the other hand, P. syringae triggers the hypersensitive response (HR), a rapid, defense-associated death of plant cells in contact with the pathogen (Klement, 1963; Klement et al., 1964; Bent, 1996; Greenberg, 1996; Dangl et al., 1996; Hammond-Kosack and Jones, 1997). In this situation, P. syringae fails to multiply to high population levels and causes no disease symptoms. Figure 1. A transmission electron microscope image of Pseudomonas syringae pv. tomato DC3000. Note that DC3000 produces polar flagella (15 nm in diameter) and a few Hrp pili (8 nm in diameter). The flagella and Hrp pili are indicated with arrows. Flagella enable ... In the late 1980s, several strains belonging to pathovars tomato, maculicola, pisi, and atropurpurea of Pseudomonas syringae were discovered to infect the model plant Arabidopsis thaliana (reviewed by Crute et al., 1994). The establishment of the Arabidopsis-P. syringae pathosystem triggered a period of highly productive research that has contributed to the elucidation of the fascinating mechanisms underlying plant recognition of pathogens, signal transduction pathways controlling plant defense responses, host susceptibility, and pathogen virulence and avirulence determinants. In this chapter we trace the discovery of this pathosystem, overview the most salient aspects of this interaction, and point out the gaps in our knowledge. At the end of this chapter we will also provide a glossary of relevant pathology-related technical terms (Appendix I), a list of people who are studying this interaction so readers can seek help if they have further questions about the Arabidopsis-P. syringae interaction (Appendix II), and several experimental procedures commonly used in the study of the Arabidopsis-P. syringae interaction (Appendix III).

Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity

Plants employ two modes of their innate immune system to resist pathogen infection. The first mode of immunity is referred to as pattern-triggered immunity (PTI) that is triggered by molecular patterns common to many types of microbes. The second mode is triggered by recognition of pathogen effectors and is called as effector-triggered immunity (ETI). At least some cases of PTI and ETI extensively share downstream signaling machinery, that is, PTI and ETI appear to be mediated by an integrated signaling network. However, activated immune responses in ETI are more prolonged and robust than those in PTI. Furthermore, network analysis has revealed that synergistic relationships among the signaling sectors are evident in PTI, which may amplify the signal, whereas compensatory relationships among the sectors dominate in ETI, explaining the robustness of ETI against genetic and pathogenic perturbations. Thus, plants seem to use a common signaling network differently in PTI and ETI.

Network properties of robust immunity in plants.

Two modes of plant immunity against biotrophic pathogens, Effector Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), are triggered by recognition of pathogen effectors and Microbe-Associated Molecular Patterns (MAMPs), respectively. Although the jasmonic acid (JA)/ethylene (ET) and salicylic acid (SA) signaling sectors are generally antagonistic and important for immunity against necrotrophic and biotrophic pathogens, respectively, their precise roles and interactions in ETI and PTI have not been clear. We constructed an Arabidopsis dde2/ein2/pad4/sid2-quadruple mutant. DDE2, EIN2, and SID2 are essential components of the JA, ET, and SA sectors, respectively. The pad4 mutation affects the SA sector and a poorly characterized sector. Although the ETI triggered by the bacterial effector AvrRpt2 (AvrRpt2-ETI) and the PTI triggered by the bacterial MAMP flg22 (flg22-PTI) were largely intact in plants with mutations in any one of these genes, they were mostly abolished in the quadruple mutant. For the purposes of this study, AvrRpt2-ETI and flg22-PTI were measured as relative growth of Pseudomonas syringae bacteria within leaves. Immunity to the necrotrophic fungal pathogen Alternaria brassicicola was also severely compromised in the quadruple mutant. Quantitative measurements of the immunity levels in all combinatorial mutants and wild type allowed us to estimate the effects of the wild-type genes and their interactions on the immunity by fitting a mixed general linear model. This signaling allocation analysis showed that, contrary to current ideas, each of the JA, ET, and SA signaling sectors can positively contribute to immunity against both biotrophic and necrotrophic pathogens. The analysis also revealed that while flg22-PTI and AvrRpt2-ETI use a highly overlapping signaling network, the way they use the common network is very different: synergistic relationships among the signaling sectors are evident in PTI, which may amplify the signal; compensatory relationships among the sectors dominate in ETI, explaining the robustness of ETI against genetic and pathogenic perturbations.

Eukaryotic Fatty Acylation Drives Plasma Membrane Targeting and Enhances Function of Several Type III Effector Proteins from Pseudomonas syringae

Bacterial pathogens of plants and animals utilize conserved type III delivery systems to traffic effector proteins into host cells. Plant innate immune systems evolved disease resistance (R) genes to recognize some type III effectors, termed avirulence (Avr) proteins. On disease-susceptible (r) plants, Avr proteins can contribute to pathogen virulence. We demonstrate that several type III effectors from Pseudomonas syringae are targeted to the host plasma membrane and that efficient membrane association enhances function. Efficient localization of three Avr proteins requires consensus myristoylation sites, and Avr proteins can be myristoylated inside the host cell. These prokaryotic type III effectors thus utilize a eukaryote-specific posttranslational modification to access the subcellular compartment where they function.

Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection.

Recognition of microbial patterns by host pattern recognition receptors is a key step in immune activation in multicellular eukaryotes. Peptidoglycans (PGNs) are major components of bacterial cell walls that possess immunity-stimulating activities in metazoans and plants. Here we show that PGN sensing and immunity to bacterial infection in Arabidopsis thaliana requires three lysin-motif (LysM) domain proteins. LYM1 and LYM3 are plasma membrane proteins that physically interact with PGNs and mediate Arabidopsis sensitivity to structurally different PGNs from Gram-negative and Gram-positive bacteria. lym1 and lym3 mutants lack PGN-induced changes in transcriptome activity patterns, but respond to fungus-derived chitin, a pattern structurally related to PGNs, in a wild-type manner. Notably, lym1, lym3, and lym3 lym1 mutant genotypes exhibit supersusceptibility to infection with virulent Pseudomonas syringae pathovar tomato DC3000. Defects in basal immunity in lym3 lym1 double mutants resemble those observed in lym1 and lym3 single mutants, suggesting that both proteins are part of the same recognition system. We further show that deletion of CERK1, a LysM receptor kinase that had previously been implicated in chitin perception and immunity to fungal infection in Arabidopsis, phenocopies defects observed in lym1 and lym3 mutants, such as peptidoglycan insensitivity and enhanced susceptibility to bacterial infection. Altogether, our findings suggest that plants share with metazoans the ability to recognize bacterial PGNs. However, as Arabidopsis LysM domain proteins LYM1, LYM3, and CERK1 form a PGN recognition system that is unrelated to metazoan PGN receptors, we propose that lineage-specific PGN perception systems have arisen through convergent evolution.

Mutational Analysis of the Arabidopsis Nucleotide Binding Site–Leucine-Rich Repeat Resistance Gene RPS2

Disease resistance proteins containing a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region compose the largest class of disease resistance proteins. These so-called NBS-LRR proteins confer resistance against a wide variety of phytopathogens. To help elucidate the mechanism by which NBS-LRR proteins recognize and transmit pathogen-derived signals, we analyzed mutant versions of the Arabidopsis NBS-LRR protein RPS2. The RPS2 gene confers resistance against Pseudomonas syringae strains carrying the avirulence gene avrRpt2. The activity of RPS2 derivatives in response to AvrRpt2 was measured by using a functional transient expression assay or by expressing the mutant proteins in transgenic plants. Directed mutagenesis revealed that the NBS and an N-terminal leucine zipper (LZ) motif were critical for RPS2 function. Mutations near the N terminus, including an LZ mutation, resulted in proteins that exhibited a dominant negative effect on wild-type RPS2. Scanning the RPS2 molecule with a small in-frame internal deletion demonstrated that RPS2 does not have a large dispensable region. Overexpression of RPS2 in the transient assay in the absence of avrRpt2 also led to an apparent resistant response, presumably a consequence of a low basal activity of RPS2. The NBS and LZ were essential for this overdose effect, whereas the entire LRR was dispensable. RPS2 interaction with a 75-kD protein (p75) required an N-terminal portion of RPS2 that is smaller than the region required for the overdose effect. These findings illuminate the pathogen recognition mechanisms common among NBS-LRR proteins.

Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae

Salicylic acid (SA)-induced defense responses are important factors during effector triggered immunity and microbe-associated molecular pattern (MAMP)-induced immunity in plants. This article presents evidence that a member of the Arabidopsis CBP60 gene family, CBP60g, contributes to MAMP-triggered SA accumulation. CBP60g is inducible by both pathogen and MAMP treatments. Pseudomonas syringae growth is enhanced in cbp60g mutants. Expression profiles of a cbp60g mutant after MAMP treatment are similar to those of sid2 and pad4, suggesting a defect in SA signaling. Accordingly, cbp60g mutants accumulate less SA when treated with the MAMP flg22 or a P. syringae hrcC strain that activates MAMP signaling. MAMP-induced production of reactive oxygen species and callose deposition are unaffected in cbp60g mutants. CBP60g is a calmodulin-binding protein with a calmodulin-binding domain located near the N-terminus. Calmodulin binding is dependent on Ca(2+). Mutations in CBP60g that abolish calmodulin binding prevent complementation of the SA production and bacterial growth defects of cbp60g mutants, indicating that calmodulin binding is essential for the function of CBP60g in defense signaling. These studies show that CBP60g constitutes a Ca(2+) link between MAMP recognition and SA accumulation that is important for resistance to P. syringae.