A dialogue-like cell communication mechanism is conserved in filamentous ascomycete fungi and mediates interspecies interactions.

In many filamentous fungi, germinating spores cooperate by fusing into supracellular structures, which develop into the mycelial colony. In the model fungus Neurospora crassa, this social behavior is mediated by an intriguing mode of communication, in which two fusing cells take turns in signal sending and receiving. Here we show that this dialogue-like cell communication mechanism is highly conserved in distantly related fungal species and mediates interspecies interactions. In mixed populations, cells of N. crassa and the phytopathogenic gray mold Botrytis cinerea coordinate their behavior over a spatial distance and establish physical contact. Subsequent cell­cell fusion is, however, restricted to germlings of the same species, indicating that species specificity of germling fusion has evolved not on the level of the signal/receptor but at subsequent levels of the fusion process. In B. cinerea, fusion and infectious growth are mutually exclusive cellular programs. Remarkably, the presence of N. crassa can reprogram this behavior and induce fusion of the gray mold on plant surfaces, potentially weakening its pathogenic potential. In a third fungal species, the nematode-trapping fungus Arthrobotrys flagrans, the conserved signaling mechanism mediates vegetative fusion within mycelial colonies but has also been repurposed for the formation of nematode-catching traps. In summary, this study identified the cell dialogue mechanism as a conserved complex trait and revealed that even distantly related fungi possess a common molecular language, which promotes cellular contact formation across species borders.

Unusual DNA-binding properties of the Arabidopsis thaliana WRKY50 transcription factor at target gene promoters.

KEY MESSAGE: WRKY50 from A. thaliana requires WT-boxes at target gene promoters for activation and binding. Based on the genome-wide prediction of WRKY50 target genes and the similarity of a WRKY50 binding site to WT-boxes in microbe-associated molecular pattern (MAMP)-responsive cis-regulatory modules (CRM), four WT-box containing CRMs from the promoter region of three WRKY50 target genes were investigated for their interaction with WRKY50. These target genes are DJ1E, WRKY30 and ATBBE4. Two of the four CRMs, one from DJ1E and one from WRKY30, were able to activate reporter gene expression in the presence of WRKY50. Activation requires the WT-boxes GGACTTTT, GGACTTTG from DJ1E and GGACTTTC from WRKY30. WRKY50 does not activate a second CRM from WRKY30 and the CRM from ATBBE4, both containing the WT-box TGACTTTT. In vitro gel-shift assays demonstrate WT-box-specific binding of the WRKY50 DNA-binding domain to all four CRMs. This work shows a high flexibility of WRKY50 binding site recognition beyond the classic W-box TTGACC/T.

A strong NF-κB p65 responsive cis-regulatory sequence from Arabidopsis thaliana interacts with WRKY40.

KEY MESSAGE: Transcription factors from mammals and plants, which play a role in innate immunity, interact with the same microbe-associated molecular pattern (MAMP)-responsive sequences from Arabidopsis thaliana. The interaction of mouse NF-κB p65 with MAMP-responsive sequences containing the core motif GACTTT of the WT-box was investigated. This revealed one sequence, derived from the promoter of the A. thaliana gene At1g76960, a gene with unknown function, to activate NF-κB p65 dependent reporter gene expression in plant cells very strongly. A bioinformatic analysis predicts three putative NF-κB p65 binding sites in this sequence and all three sites are required for reporter gene activation and binding. The sequence is one of the weakest MAMP-responsive sequences previously isolated, but the introduction of a GCC-box increases its MAMP responsivity in combination with upstream WT-box sequences. Although a bioinformatic analysis of the unmutated cis-sequence only predicts NF-κB p65 binding, A. thaliana WRKY40 was selected in a yeast one-hybrid screen. WRKY40, which is a transcriptional repressor, requires the sequence TTTTCTA for direct binding. This sequence is similar to the WK-box TTTTCCAC, previously shown to interact with tobacco NtWRKY12. In summary, this work supports the similarity in binding site recognition between NF-κB and WRKY factors.

Transcription factors involved in basal immunity in mammals and plants interact with the same MAMP-responsive cis-sequence from Arabidopsis thaliana.

KEY MESSAGE: WRKY and NF-κB transcription factors, involved in innate immunity in plants and mammals, interact with the same cis-sequence. Novel microbe-associated molecular pattern (MAMP)-responsive cis-sequences, designated type II WT-boxes, are required for flg22-responsive gene expression in Arabidopsis thaliana protoplasts. While type I WT-boxes like TGACTTTT and CGACTTTT interact with WRKY transcription factors (TFs), the question remained which TFs bind to the type II WT-boxes GGACTTTC, GGACTTTT, and GGACTTTG. Surprisingly, a bioinformatic analysis predicts mouse (Mus musculus) NF-κB p65 as a TF interacting with type II WT-boxes. NF-κB p65, like WRKY factors in plants, plays a role in innate immunity in mammals. Therefore, the interaction of NF-κB p65 with type II WT-boxes was tested experimentally. NF-κB p65 requires the WT-boxes GGACTTTC, GGACTTTT, and GGACTTTG for activating reporter gene expression in plant cells. NF-κB p65 directly binds to WT-box containing synthetic promoters in vitro and requires the WT-box for binding. Earlier studies indicate that the sequence GGACTTTC is also required for WRKY26 mediated reporter gene activation. Here it is shown that WRKY26, like NF-κB p65, binds to the sequence GGACTTTC. Consistent with other recent studies, type II WT boxes are WRKY binding sites and the distinction between type I and type II no longer applies.

Combinatorial requirement of W- and WT-boxes in microbe-associated molecular pattern-responsive synthetic promoters.

KEY MESSAGE: The WT-box GGACTTTC belongs to a novel class of MAMP-responsive cis-regulatory sequences that are part of combinatorial elements. Microbe-associated molecular pattern (MAMP)-responsive synthetic promoters were generated with two cis-regulatory modules (CRM1 and CRM2) from the Arabidopsis thaliana WRKY30 promoter. Both modules harbour two W-boxes and one WT-box. Mutation analysis of the synthetic promoters and transient gene expression analysis in parsley protoplasts underline the importance of the W- and WT-boxes for MAMP-responsive gene expression and reveal the combinatorial requirement of at least two boxes for full MAMP responsivity. In the context of the native promoter, CRM1 is required for MAMP responsivity, while CRM2 alone is not sufficient. Yeast one-hybrid screenings using CRM1 with a transcription factor (TF) only prey library select only WRKY factors. Selection of WRKY26, 40, 41, and 70 requires the W-boxes. The WT-box is also required for selection of WRKY26 and 41 in yeast. In plant cells, WRKY26, 40, and 41 act as repressors of MAMP-responsive gene expression, whereas WRKY70 is an activator. To investigate whether the WT-box is also required for WRKY26 and 41 mediated gene expression in plant cells, both were converted into transcriptional activators by adding the GAL4 activating domain (AD). In contrast to yeast, transient gene expression in parsley protoplasts shows that only the W-boxes from CRM1 are required for WRKY41AD-activated reporter gene activity but not the WT-box. In addition, WRKY70-activated reporter gene activity in parsley cells does not require the WT-box of CRM1. The results demonstrate the importance of the WT-box as a new cis-regulatory sequence for MAMP-responsive gene expression. Based on these and earlier results, two types of WT-boxes are proposed.

Analysis of Microbe-Associated Molecular Pattern-Responsive Synthetic Promoters with the Parsley Protoplast System.

Plants recognize pathogens by microbe-associated molecular patterns (MAMPs) and subsequently induce an immune response. The regulation of gene expression during the immune response depends largely on cis-sequences conserved in promoters of MAMP-responsive genes. These cis-sequences can be analyzed by constructing synthetic promoters linked to a reporter gene and by testing these constructs in transient expression systems. Here, the use of the parsley (Petroselinum crispum) protoplast system for analyzing MAMP-responsive synthetic promoters is described. The synthetic promoter consists of four copies of a potential MAMP-responsive cis-sequence cloned upstream of a minimal promoter and the uidA reporter gene. The reporter plasmid contains a second reporter gene, which is constitutively expressed and hence eliminates the requirement of a second plasmid used as a transformation control. The reporter plasmid is transformed into parsley protoplasts that are elicited by the MAMP Pep25. The MAMP responsiveness is validated by comparing the reporter gene activity from MAMP-treated and untreated cells and by normalizing reporter gene activity using the constitutively expressed reporter gene.

Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter.

Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast one-hybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA.