Thomas Kroj

The Transcription Factors WRKY11 and WRKY17 Act as Negative Regulators of Basal Resistance in Arabidopsis thaliana

Transcription factors are believed to play a pivotal role in the activation and fine-tuning of plant defense responses, but little is known about the exact function of individual transcription factors in this process. We analyzed the role of the IId subfamily of WRKY transcription factors in the regulation of basal resistance to Pseudomonas syringae pv tomato (Pst). The expression of four members of the subfamily was induced upon challenge with virulent and avirulent strains of Pst. Mutant analyses revealed that loss of WRKY11 function increased resistance toward avirulent and virulent Pst strains and that resistance was further enhanced in wrky11 wrky17 double mutant plants. Thus, WRKY11 and WRKY17 act as negative regulators of basal resistance to Pst. Genome-wide expression analysis and expression studies of selected genes in single and double mutants demonstrated that both transcription factors modulate transcriptional changes in response to pathogen challenge. Depending on the target gene, WRKY11 and WRKY17 act either specifically or in a partially redundant manner. We demonstrate complex cross-regulation within the IId WRKY subfamily and provide evidence that both WRKY transcription factors are involved in the regulation of Pst-induced jasmonic acid–dependent responses. These results provide genetic evidence for the importance of WRKY11 and WRKY17 in plant defense.

Regulation of storage protein gene expression in Arabidopsis.

The expression of seed storage proteins is under tight developmental regulation and represents a powerful model system to study the regulation of gene expression during plant development. In this study, we show that three homologous B3 type transcription factors regulate the model storage protein gene, At2S3, via two distinct mechanisms: FUSCA3 (FUS3) and LEAFY COTYLEDON2 (LEC2) activate the At2S3 promoter in yeast suggesting that they regulate At2S3 by directly binding its promoter; ABSCISIC ACID INSENSITIVE3 (ABI3), however, appears to act more indirectly on At2S3, possibly as a cofactor in an activation complex. In accordance with this, FUS3 and LEC2 were found to act in a partially redundant manner and differently from ABI3 in planta: At2S3 expression is reduced to variable and sometimes only moderate extent in fus3 and lec2 single mutants but is completely abolished in the lec2 fus3 double mutant. In addition, we found that FUS3 and LEC2 expression patterns, together with an unsuspected regulation of FUS3 by LEC2, enable us to explain the intriguing expression pattern of At2S3 in lec2 or fus3 single mutants. Based on these results, we present a model of At2S3 regulation and discuss its implications for other aspects of seed maturation.

The Rice Resistance Protein Pair RGA4/RGA5 Recognizes the Magnaporthe oryzae Effectors AVR-Pia and AVR1-CO39 by Direct Binding

This work shows that the rice NB-LRR protein pair, RGA4 and RGA5-A, has a dual recognition specificity and detects the Magnaporthe oryzae effectors AVR1-CO39 and AVR-Pia, which have unrelated sequences. Recognition seems to be mediated by direct binding of the Avr proteins to a novel non-LRR domain of RGA5-A also present in the Avr binding domain of the rice resistance protein Pik-1. Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer–fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.

A novel conserved mechanism for plant NLR protein pairs: the ‘integrated decoy’ hypothesis

Plant immunity is often triggered by the specific recognition of pathogen effectors by intracellular nucleotide-binding, leucine-rich repeat receptors (NLR). Plant NLRs contain an N-terminal signaling domain that is mostly represented by either a Toll-interleukin1 receptor (TIR) domain or a coiled coil (CC) domain. In many cases, single NLR proteins are sufficient for both effector recognition and signaling activation. However, many paired NLRs have now been identified where both proteins are required to confer resistance to pathogens. Recent detailed studies on the Arabidopsis thaliana TIR-NLR pair RRS1 and RPS4 and on the rice CC-NLR pair RGA4 and RGA5 have revealed for the first time how such protein pairs function together. In both cases, the paired partners interact physically to form a hetero-complex receptor in which each partner plays distinct roles in effector recognition or signaling activation, highlighting a conserved mode of action of NLR pairs across both monocotyledonous and dicotyledonous plants. We also describe an “integrated decoy” model for the function of these receptor complexes. In this model, a plant protein targeted by an effector has been duplicated and fused to one member of the NLR pair, where it acts as a bait to trigger defense signaling by the second NLR upon effector binding. This mechanism may be common to many other plant NLR pairs.

Analysis of an activated ABI5 allele using a new selection method for transgenic Arabidopsis seeds.

The Arabidopsis abscisic acid (ABA) insensitive (ABI)5 transcription factor participates in the ABA‐dependent induction of late embryogenesis abundant (LEA) genes in the final stages of seed development. We tested whether the VP16 transcriptional activation domain is sufficient to provide ABI5 with the ability to activate the AtEm LEA genes in vegetative tissues. We took advantage of a new transgenic seed selection assay based on green fluorescent protein (GFP) fluorescence and found that VP16‐ABI5 triggered growth retardation and ABA‐independent induction of AtEm1 in seedlings. These results indicate that ABI5 activation potential is a limiting step and might be a target for ABA signaling.

Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis

The deposition of lignin during plant-pathogen interactions is thought to play a role in plant defence. However, the function of lignification genes in plant disease resistance is poorly understood. In this article, we provide genetic evidence that the primary genes involved in lignin biosynthesis in Arabidopsis, CAD-C and CAD-D, act as essential components of defence to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv. tomato, possibly through the salicylic acid defence pathway. Thus, in contrast with cellulose synthesis, whose alteration leads to an increase in disease resistance, alteration of the cell wall lignin content leads directly or indirectly to defects in some defence components.

VASCULAR ASSOCIATED DEATH1, a Novel GRAM Domain–Containing Protein, Is a Regulator of Cell Death and Defense Responses in Vascular Tissues

The hypersensitive response (HR) is a programmed cell death that is commonly associated with plant disease resistance. A novel lesion mimic mutant, vad1 (for vascular associated death1), that exhibits light conditional appearance of propagative HR-like lesions along the vascular system was identified. Lesion formation is associated with expression of defense genes, production of high levels of salicylic acid (SA), and increased resistance to virulent and avirulent strains of Pseudomonas syringae pv tomato. Analyses of the progeny from crosses between vad1 plants and either nahG transgenic plants, sid1, nonexpressor of PR1 (npr1), enhanced disease susceptibility1 (eds1), or non-race specific disease resistance1 (ndr1) mutants, revealed the vad1 cell death phenotype to be dependent on SA biosynthesis but NPR1 independent; in addition, both EDS1 and NDR1 are necessary for the proper timing and amplification of cell death as well as for increased resistance to Pseudomonas strains. VAD1 encodes a novel putative membrane-associated protein containing a GRAM domain, a lipid or protein binding signaling domain, and is expressed in response to pathogen infection at the vicinity of the hypersensitive lesions. VAD1 might thus represent a new potential function in cell death control associated with cells in the vicinity of vascular bundles.

The NB‐LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance

Plant resistance proteins of the class of nucleotide‐binding and leucine‐rich repeat domain proteins (NB‐LRRs) are immune sensors which recognize pathogen‐derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB‐LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR‐independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR‐Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo‐ and hetero‐complexes and interact through their coiled‐coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR‐Pia, neither RGA4 nor RGA5 is re‐localized to the nucleus. These results establish a model for the interaction of hetero‐pairs of NB‐LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.

Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

Summary Plant immune receptors of the class of nucleotide‐binding and leucine‐rich repeat domain (NLR) proteins can contain additional domains besides canonical NB‐ARC (nucleotide‐binding adaptor shared by APAF‐1, R proteins, and CED‐4 (NB‐ARC)) and leucine‐rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger–BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over‐expression and knock‐out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in‐depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system.

AvrACXcc8004, a Type III Effector with a Leucine-Rich Repeat Domain from Xanthomonas campestris Pathovar campestris Confers Avirulence in Vascular Tissues of Arabidopsis thaliana Ecotype Col-0

Xanthomonas campestris pathovar campestris causes black rot, a vascular disease on cruciferous plants, including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 encodes a protein containing leucine-rich repeats (LRRs) and appears to be restricted to strains of X. campestris pv. campestris. LRRs are found in a number of type III-secreted effectors in plant and animal pathogens. These prompted us to investigate the role of the XC1553 gene in the interaction between X. campestris pv. campestris and A. thaliana. Translocation assays using the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter revealed that XC1553 is a type III effector. Infiltration of Arabidopsis leaf mesophyll with bacterial suspensions showed no differences between the wild-type strain and an XC1553 gene mutant; both strains induced disease symptoms on Kashmir and Col-0 ecotypes. However, a clear difference was observed when bacteria were introduced into the vascular system by piercing the central vein of leaves. In this case, the wild-type strain 8004 caused disease on the Kashmir ecotype, but not on ecotype Col-0; the XC1553 gene mutant became virulent on the Col-0 ecotype and still induced disease on the Kashmir ecotype. Altogether, these data show that the XC1553 gene, which was renamed avrAC(Xcc8004), functions as an avirulence gene whose product seems to be recognized in vascular tissues.