MicroRNAs constitute an additional layer in plant response to simultaneous bio- and abiotic stresses as exemplified by UV-B radiation and flg22-treatment on Arabidopsis thaliana.

Plants are confronted with various environmental stresses and develop sophisticated adaptive mechanisms. Our previous work demonstrated that the crosstalk of flg22 and ultraviolet (UV)-B-induced signalling cascades reprograms the expression of flavonol pathway genes (FPGs), benefiting plant defence responses. Although several transcription factors have been identified to be involved in this crosstalk, the underlying mechanism is largely unclear. Here, we analyzed microRNAs (miRNAs) and identified 126, 129 and 113 miRNAs with altered abundances compared to untreated control in flg22-, UV-B- and flg22/UV-B-treated seedlings, respectively. Two distinct modules were identified: The first consists of 10 miRNAs repressed by UV-B but up-regulated by flg22, and the second with five miRNAs repressed by flg22 but up-regulated by UV-B. In Arabidopsis, the knockdown of miR858a, a representative of module I, increased the abundance of CHS (a marker gene for FPGs), whereas its overexpression reduced CHS. Conversely, knockout of miR164b from module II decreased CHS and its overexpression increased CHS transcript levels. These data suggest a decisive role of miRNAs in the crosstalk. In the next, we described the interaction between miR858a and its target MYB111 (a positive regulator of FPGs) from module I in detail. We showed that MYB111 was profoundly post-transcriptionally regulated by miR858a during the crosstalk, whose expression was specifically but antagonistically controlled by UVR8- and FLS2-mediated signallings. Moreover, transcriptional monitoring using the GUS reporter gene demonstrates that miRNA-mediated posttranscriptional regulation is the main driving force in reprogramming the expression of FPGs and regulates plant adaptation to multiple concurrent environmental stresses.

RNAseq-Based Working Model for Transcriptional Regulation of Crosstalk between Simultaneous Abiotic UV-B and Biotic Stresses in Plants.

Plants adjust their secondary metabolism by altering the expression of corresponding genes to cope with both abiotic and biotic stresses. In the case of UV-B radiation, plants produce protective flavonoids; however, this reaction is impeded during pattern-triggered immunity (PTI) induced by pathogens. Pathogen attack can be mimicked by the application of microbial associated molecular patterns (e.g., flg22) to study crosstalk between PTI and UV-B-induced signaling pathways. Switching from Arabidopsis cell cultures to in planta studies, we analyzed whole transcriptome changes to gain a deeper insight into crosstalk regulation. We performed a comparative transcriptomic analysis by RNAseq with four distinct mRNA libraries and identified 10778, 13620, and 11294 genes, which were differentially expressed after flg22, UV-B, and stress co-treatment, respectively. Focusing on genes being either co-regulated with the UV-B inducible marker gene chalcone synthase CHS or the flg22 inducible marker gene FRK1 identified a large set of transcription factors from diverse families, such as MYB, WRKY, or NAC. These data provide a global view of transcriptomic reprogramming during this crosstalk and constitute a valuable dataset for further deciphering the underlying regulatory mechanism(s), which appear to be much more complex than previously anticipated. The possible involvement of MBW complexes in this context is discussed.

MicroRNA840 (MIR840) accelerates leaf senescence by targeting the overlapping 3'UTRs of PPR and WHIRLY3 in Arabidopsis thaliana.

MicroRNAs negatively regulate gene expression by promoting target mRNA cleavage and/or impairing its translation, thereby playing a crucial role in plant development and environmental stress responses. In Arabidopsis, the MIR840 gene is located within the overlapping 3'UTR of the PPR and WHIRLY3 (WHY3) genes, both being predicted targets of miR840* and miR840, the short maturation products of MIR840. Gain- and loss-of-function of MIR840 in Arabidopsis resulted in opposite senescence phenotypes. The highest expression levels of the MIR840 precursor transcript pre-miR840 were observed at senescence initiation, and pre-miR840 expression is significantly correlated with a reduction in PPR, but not WHY3, transcript levels. Although a reduction of transcript level of PPR, but not WHY3 transcript levels were not significantly affected by MIR840 overexpression, its protein levels were strongly reduced. Mutating the cleavage sites or replacing the target sequences abolishes the miR840*/miR840-mediated degradation of PPR transcripts and accumulation of WHY3 protein. In support for this, concurrent knockdown of both PPR and WHY3 in wild-type plants resulted in a senescence phenotype resembling that of the MIR840-overexpressing plant. This indicates that both PRR and WHY3 are targets in the MIR840-mediated senescence pathway. Moreover, single knockout mutants of PPR and WHY3 show a convergent upregulated subset of senescence-associated genes, which are also found among those induced by MIR840 overexpression. Our data provide evidence for a regulatory role of MIR840 in plant senescence.

Dual-Localized WHIRLY1 Affects Salicylic Acid Biosynthesis via Coordination of ISOCHORISMATE SYNTHASE1, PHENYLALANINE AMMONIA LYASE1, and S-ADENOSYL-L-METHIONINE-DEPENDENT METHYLTRANSFERASE1.

Salicylic acid (SA) influences developmental senescence and is spatiotemporally controlled by various mechanisms, including biosynthesis, transport, and conjugate formation. Altered localization of Arabidopsis WHIRLY1 (WHY1), a repressor of leaf natural senescence, in the nucleus or chloroplast causes a perturbation in SA homeostasis, resulting in adverse plant senescence phenotypes. WHY1 loss-of-function mutation resulted in SA peaking 5 d earlier compared to wild-type plants, which accumulated SA at 42 d after germination. SA accumulation coincided with an early leaf-senescence phenotype, which could be prevented by ectopic expression of the nuclear WHY1 isoform (nWHY1). However, expressing the plastid WHY1 isoform (pWHY1) greatly enhanced cellular SA levels. Transcriptome analysis in the WHY1 loss-of-function mutant background following expression of either pWHY1 or nWHY1 indicated that hormone metabolism-related genes were most significantly altered. The pWHY1 isoform predominantly affected stress-related gene expression, whereas nWHY1 primarily controlled developmental gene expression. Chromatin immunoprecipitation-quantitative PCR assays indicated that nWHY1 directly binds to the promoter region of isochorismate synthase1 (ICS1), thus activating its expression at later developmental stages, but that it indirectly activates S-adenosyl- l -Met-dependent methyltransferase1 (BSMT1) expression via ethylene response factor 109 (ERF109). Moreover, nWHY1 repressed expression of Phe ammonia lyase-encoding gene (PAL1) via R2R3-MYB member 15 (MYB15) during the early stages of development. Interestingly, rising SA levels exerted a feedback effect by inducing nWHY1 modification and pWHY1 accumulation. Thus, the alteration of WHY1 organelle isoforms and the feedback of SA are involved in a circularly integrated regulatory network during developmental or stress-induced senescence in Arabidopsis.

Applications of CRISPR/Cas to Improve Crop Disease Resistance: Beyond Inactivation of Susceptibility Factors.

Current crop production systems are prone to increasing pathogen pressure. Fundamental understanding of molecular plant-pathogen interactions, the availability of crop and pathogen genomic information, as well as emerging genome editing permits a novel approach for breeding of crop disease resistance. We describe here strategies to identify new targets for resistance breeding with focus on interruption of the compatible plant-pathogen interaction by CRISPR/Cas-mediated genome editing. Basically, crop genome editing can be applied in several ways to achieve this goal. The most common approach focuses on the "simple" knockout by non-homologous end joining repair of plant susceptibility factors required for efficient host colonization. However, genome re-writing via homology-directed repair or base editing can also prevent host manipulation by changing the targets of pathogen-derived effectors or molecules beyond recognition, which also decreases plant susceptibility. We conclude that genome editing by CRISPR/Cas will become increasingly indispensable to generate in relatively short time beneficial resistance traits in crops to meet upcoming challenges.

Phytohormone crosstalk in the host-Verticillium interaction.

Functional genomics can be applied to shed light on the Brassica napus - Verticillium interaction. RNAseq data indicated already that abscisic acid (ABA) is apparently involved in the early oilseed rape response to fungal infection with Verticillium longisporum isolate 43 (Vl43). A calreticulin (CRT1a) was identified as novel susceptibility factor for Vl43 infecting both Arabidopsis and oilseed rape. CRT1a is involved in calcium homeostasis and contributes in the endoplasmatic reticulum to the unfolded protein response. The latter function could either affect the correct folding of other susceptibility factors or of negative regulators in ethylene (ET) signaling. Which CRT1a function is affected in the mutants is currently unknown, but both hypotheses can explain that crt1a loss-of-function mutants display increased resistance to V. longisporum and enhanced expression of ethylene signaling related genes. This indicates that besides other phytohormones such as ABA or salicylic acid (SA) also ET plays a critical role in the plant-Verticillium interaction, which might be exploited to improve plant resistance.

Loss of function of CRT1a (calreticulin) reduces plant susceptibility to Verticillium longisporum in both Arabidopsis thaliana and oilseed rape (Brassica napus).

Brassica napus is highly susceptible towards Verticillium longisporum (Vl43) with no effective genetic resistance. It is believed that the fungus reprogrammes plant physiological processes by up-regulation of so-called susceptibility factors to establish a compatible interaction. By transcriptome analysis, we identified genes, which were activated/up-regulated in rapeseed after Vl43 infection. To test whether one of these genes is functionally involved in the infection process and loss of function would lead to decreased susceptibility, we firstly challenged KO lines of corresponding Arabidopsis orthologs with Vl43 and compared them with wild-type plants. Here, we report that the KO of AtCRT1a results in drastically reduced susceptibility of plants to Vl43. To prove crt1a mutation also decreases susceptibility in B. napus, we identified 10 mutations in a TILLING population. Three T3 mutants displayed increased resistance as compared to the wild type. To validate the results, we generated CRISPR/Cas-induced BnCRT1a mutants, challenged T2 plants with Vl43 and observed an overall reduced susceptibility in 3 out of 4 independent lines. Genotyping by allele-specific sequencing suggests a major effect of mutations in the CRT1a A-genome copy, while the C-genome copy appears to have no significant impact on plant susceptibility when challenged with Vl43. As revealed by transcript analysis, the loss of function of CRT1a results in activation of the ethylene signalling pathway, which may contribute to reduced susceptibility. Furthermore, this study demonstrates a novel strategy with great potential to improve plant disease resistance.

Suppression of abscisic acid biosynthesis at the early infection stage of Verticillium longisporum in oilseed rape (Brassica napus).

Verticillium longisporum infects oilseed rape (Brassica napus) and Arabidopsis thaliana. To investigate the early response of oilseed rape to the fungal infection, we determined transcriptomic changes in oilseed rape roots at 6 days post-inoculation (dpi) by RNA-Seq analysis, in which non-infected roots served as a control. Strikingly, a subset of genes involved in abscisic acid (ABA) biosynthesis was found to be down-regulated and the ABA level was accordingly attenuated in 6 dpi oilseed rape as compared with the control. Gene expression analysis revealed that this was mainly attributed to the suppression of BnNCED3-mediated ABA biosynthesis, involving, for example, BnWRKY57. However, this down-regulation of ABA biosynthesis could not be observed in infected Arabidopsis roots. Arabidopsis ABA- defective mutants nced3 and aao3 displayed pronounced tolerance to the fungal infection with delayed and impeded symptom development, even though fungal colonization was not affected in both mutants. These data suggest that ABA appears to be required for full susceptibility of Arabidopsis to the fungal infection. Furthermore, we found that in both 6 dpi oilseed rape and the Arabidopsis nced3 mutant, the salicylic acid (SA) signalling pathway was induced while the jasmonic acid (JA)/ethylene (ET) signalling pathway was concomitantly mitigated. Following these data, we conclude that in oilseed rape the V. longisporum infection triggers a host-specific suppression of the NCED3-mediated ABA biosynthesis, consequently increasing plant tolerance to the fungal infection. We believe that this might be part of the virulence strategy of V. longisporum to initiate/establish a long-lasting compatible interaction with oilseed rape (coexistence), which appears to be different from the infection process in Arabidopsis.

Suppression of UV-B stress induced flavonoids by biotic stress: Is there reciprocal crosstalk?

Plants respond to abiotic UV-B stress with enhanced expression of genes for flavonoid production, especially the key-enzyme chalcone synthase (CHS). Some flavonoids are antioxidative, antimicrobial and/or UV-B protective secondary metabolites. However, when plants are challenged with concomitant biotic stress (simulated e.g. by the bacterial peptide flg22, which induces MAMP triggered immunity, MTI), the production of flavonoids is strongly suppressed in both Arabidopsis thaliana cell cultures and plants. On the other hand, flg22 induces the production of defense related compounds, such as the phytoalexin scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread within the host tissue. Since all these metabolites require the precursor phenylalanine for their production, suppression of the flavonoid production appears to allow the plant to focus its secondary metabolism on the production of pathogen defense related compounds during MTI. Interestingly, several flavonoids have been reported to display anti-microbial activities. For example, the plant flavonoid phloretin targets the Pseudomonas syringae virulence factors flagella and type 3 secretion system. That is, suppression of flavonoid synthesis during MTI might have also negative side-effects on the pathogen defense. To clarify this issue, we deployed an Arabidopsis flavonoid mutant and obtained genetic evidence that flavonoids indeed contribute to ward off the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Finally, we show that UV-B attenuates expression of the flg22 receptor FLS2, indicating that there is negative and reciprocal interaction between this abiotic stress and the plant-pathogen defense responses.

Investigation of the crosstalk between the flg22 and the UV-B-induced flavonol pathway in Arabidopsis thaliana seedlings.

In Arabidopsis cell culture, we have shown that flavonol metabolite accumulation depends on expression of the flavonol pathway genes (FPGs), which are up-regulated by UV-B irradiation but repressed during microbe-associated molecular pattern (MAMP) -triggered immunity (MTI) induced by the bacterial elicitor flg22. The suppression of flavonoid production during MTI is believed to allow the plant focusing its metabolism on the pathogen defense by directing phenylalanine resources from UV-B protective flavonol production towards production of phytoalexins and cell wall fortification by lignin incorporation. Here, we show that UV-B-induced FPGs are also suppressed by flg22 in Arabidopsis seedlings, demonstrating that this kind of 'crosstalk' is fully functional in planta. We believe that this system based on seedlings of the model plant Arabidopsis thaliana constitutes a valuable tool for further dissection of the underlying molecular mechanism, for example, by deploying gain/loss-of-function mutants. Furthermore, we observed some differences in the expression patterns of MYB transcription factors (TFs) as compared to data from the cell culture system. The new data suggest that in planta the TF MYB111 might play a more dominant role than the TF MYB12, which was strongly regulated in cell cultures. Thus, we can present an updated working model how this crosstalk might function.

The interplay of transcription factors in suppression of UV-B induced flavonol accumulation by flg22.

Biotic stress can be mimicked by application of elicitors, which comprise of microbe-associated molecular patterns (MAMPs). Treatment of plant cell cultures with MAMPs such as flg22 suppressed the expression of UV-B-induced flavonol pathway genes (FPGs) in parsley, carrot and Arabidopsis. This is thought to allow the plant focusing its secondary metabolism on the pathogen defense during MAMP-triggered immunity (MTI). Recently we reported that this suppression also depends on prevention of histone 3 acetylation at lysine 9 (H3K9ac), a hallmark for gene activation. Here we describe a possible regulation between UV-B and flg22 signaling cascades, and the interplay of MYB and WRKY transcription factors in regulating the expression of the FPGs.

Suppression of UV-B stress responses by flg22 is regulated at the chromatin level via histone modification.

Genes of the flavonol pathway are activated by UV-B, but suppressed by concomitant flg22 application in Arabidopsis. Analysis at the metabolite level suggested that this regulation allows the plant to focus its secondary metabolism on the plant defence towards pathogen attack. We now demonstrate by chromatin immunoprecipitation followed by quantitative PCR, that this antagonistic gene regulation is mediated at the chromatin level by differential regulation of histone 3 lysine 9 acetylation (H3K9ac), which is a hallmark for gene activation. Since H3K9ac levels were altered at least at four independent gene loci, namely, chalcone synthase, chalcone-flavone isomerase, flavanone 3-hydroxylase and the positive regulator MYB12, which correlates with the observed gene activation/suppression reported previously, it appears that this process is mediated by chromatin remodelling. Since suppression of H3K9ac prevents gene expression, we conclude H3K9ac is rather cause than consequence of gene activation. This finding allows us also to extend our working model, involving the two opposing MYB transcription factors of the flavonol pathway, MYB12 (being UV-B-activated and flg22-suppressed) and MYB4 (a negative regulator, which is activated by both flg22 and UV-B stress).

Crosstalk between abiotic ultraviolet-B stress and biotic (flg22) stress signalling in Arabidopsis prevents flavonol accumulation in favor of pathogen defence compound production.

Plants respond to both abiotic and biotic stresses with alterations in the expression of genes required to produce protective metabolites. Sometimes plants can be challenged with different stresses simultaneously and as they cannot evade from this situation, priorities have to be set to deal with the most urgent threat. The abiotic stress ultraviolet-B (UV-B) light induces the production of UV-protective flavonols in Arabidopsis Col-0 cell suspension cultures and this accumulation is attenuated by concurrent application of the bacterial elicitor flg22 (simulating biotic stress). This inhibition correlates with strong suppression of the flavonol biosynthesis genes. In parallel, flg22 induces the production of defence-related compounds, such as the phytoalexins, camalexin and scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread. This correlated positively with flg22-mediated expression of enzymes for lignin, scopoletin and camalexin production. As flavonols, lignin and scopoletin are all derived from phenylalanine, it appears that the plant focuses the metabolism on production of scopoletin and lignin at the expense of flavonol production. Furthermore, it appears that this crosstalk involves antagonistic regulation of two opposing MYB transcription factors, the positive regulator of the flavonol pathway MYB12 (UV-B-induced and flg22-suppressed) and the negative regulator MYB4 (UV-B- and flg22-induced).

Genomic structure of the NtPDR1 gene, harboring the two miniature inverted-repeat transposable elements, NtToya1 and NtStowaway101.

Here we report the genomic structure including the promoter sequence and coding region of NtPDR1 (Nicotiana tabacum Pleiotropic Drug Resistance 1), which is an elicitor-responsive gene encoding an ATP binding cassette (ABC) transporter that might be involved in the defense response in tobacco, as we reported recently. The NtPDR1 gene consists of 20 exons and 19 introns. Among the introns, the first and fifth are much larger than the others and harbor typical miniature inverted-repeat transposable elements (MITEs). One of the MITE elements in the first intron, termed NtToya1, belongs to the Toya family that was recently described in rice, while the other element in the fifth intron, termed NtStowaway101, shows high homology with the Stowaway elements of the IS630-Tc1-mariner family. Many of the genes we found to harbor Toya and Stowaway elements in Nicotiana species by BLAST search are also involved in stress responses or plant-pathogen interactions. The existence of putative cis-elements (a GCC box, three W boxes, and several JA-responsive elements) in the promoter region supports our previous finding that this gene is strongly inducible by elicitation and methyljasmonate, and that this ABC transporter might be essential for plant defense responses. Furthermore, Southern blot analysis and PCR amplification of the introns harboring the MITE-like elements from genomic DNA of three Nicotiana species suggests that NtPDR1 originated from N. sylvestris.