Proteomics and functional analyses of <Emphasis Type="Italic">Arabidopsis</Emphasis> nitrilases involved in the defense response to microbial pathogens

Main conclusion

Proteomics and functional analyses of the Arabidopsis – Pseudomonas syringae pv. tomato interactions reveal that Arabidopsis nitrilases are required for plant defense and R gene-mediated resistant responses to microbial pathogens. A high-throughput in planta proteome screen has identified Arabidopsis nitrilase 2 (AtNIT2), which was de novo-induced by Pseudomonas syringae pv. tomato (Pst) infection. The AtNIT2, AtNIT3, and AtNIT4 genes, but not AtNIT1, were distinctly induced in Arabidopsis leaves by Pst infection. Notably, avirulent Pst DC3000 (avrRpt2) infection led to significant induction of AtNIT2 and AtNIT4 in leaves. Pst DC3000 and Pst DC3000 (avrRpt2) significantly grew well in leaves of nitrilase transgenic (nit2i-2) and mutant (nit1-1 and nit3-1) lines compared to the wild-type leaves. In contrast, NIT2 overexpression in nit2 mutants led to significantly high growth of the two Pst strains in leaves. The nitrilase transgenic and mutant lines exhibited enhanced susceptibility to Hyaloperonospora arabidopsidis infection. The nit2 mutation enhanced Pst DC3000 (avrRpt2) growth in salicylic acid (SA)-deficient NahG transgenic and sid2 and npr1 mutant lines. Infection with Pst DC3000 or Pst DC3000 (avrRpt2) induced lower levels of indole-3-acetic acid (IAA) in nit2i and nit2i NahG plants than in wild-type plants, but did not alter the IAA level in NahG transgenic plants. This suggests that Arabidopsis nitrilase 2 is involved in IAA signaling of defense and R gene-mediated resistance responses to Pst infection. Quantification of SA in these transgenic and mutant plants demonstrates that Arabidopsis nitrilase 2 is not required for SA-mediated defense response to the virulent Pst DC3000 but regulates SA-mediated resistance to the avirulent Pst DC3000 (avrRpt2). These results collectively suggest that Arabidopsis nitrilase genes are involved in plant defense and R gene-mediated resistant responses to microbial pathogens.

     

Interaction of jasmonic acid and blue light in the regulation of arabidopsis morphogenesis

The effects of blue light (BL) and jasmonic acid (JA) on morphogenesis of Arabidopsis thaliana (L.) Heynh seedlings of genotypes Col and Ler and their mutants, namely, axr1-3 and jar1-1 mutants resistant to IAA and JA, respectively, and a CRY1 photoreceptor-deficient mutant hy4 were studied. Both 1 μM JA and BL exposure retarded hypocotyl growth of Ler, Col, and jar1-1 seedlings, whereas JA had no effect on hypocotyl growth of axr1-3, but the suppression of hypocotyl growth of this mutant by BL was even more noticeable than that of Ler, Col, and jar1-1. JA and BL applied simultaneously inhibited hypocotyl growth of axr1-3 and especially of Ler, Col, and jar1-1 more than either of factors applied separately. The hy4 mutant did not respond to BL, whereas JA stimulated its hypocotyl growth. JA did not change the cotyledon size of Col, axr1-3, and jar1-1 and reduced the cotyledon size of Ler and hy4. BL enhanced the cotyledon growth of all wild-type and mutant plants used in the study. The cotyledon sizes of all plants except Ler were also increased when JA and BL were applied together. Some of the growth responses correlated with the endogenous IAA and ABA contents. Thus, for example, the hypocotyl and cotyledon growth retardation of Ler seedlings in the presence of JA correlated with a reduced level of free IAA and a considerable increase in the free ABA level in plants grown both in darkness and in BL. Under other growth conditions, no correlation between the endogenous IAA and ABA levels and A. thaliana seedling growth was noted. The interaction between the signal transduction pathways triggered by BL and JA at the early stages of arabidopsis morphogenesis is discussed on the basis of Col, Ler, axr1-3, and jar1-1 hypocotyl growth responses.     

Content of Osmolytes and Flavonoids under Salt Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Plants Defective in Jasmonate Signaling

The effects of the salt stress (200 mM NaCl) and exogenous jasmonic acid (JA) on levels of osmolytes and flavonoids in leaves of four-week-old Arabidopsis thaliana L. plants of the wild-type (WT) Columbia-0 (Col-0) and the mutant jin1 (jasmonate insensitive 1) with impaired jasmonate signaling were studied. The increase in proline content caused by the salt stress was higher in the Col-0 plants than in the mutant jin1. This difference was especially marked if the plants had been pretreated with exogenous 0.1 μM JA. The sugar content increased in response to the salt stress in the JA-treated WT plants but decreased in the jin1 mutant. Treatment with JA of the WT plants but not mutant defective in jasmonate signaling also enhanced the levels of anthocyanins and flavonoids absorbed in UV-B range in leaves. The presence of JA increased salinity resistance of the Col-0 plants, since the accumulation of lipid peroxidation products and growth inhibition caused by NaCl were less pronounced. Under salt stress, JA almost did not render a positive effect on the jin1 plants. It is concluded that the protein JIN1/MYC2 is involved in control of protective systems under salt stress.     

Cytoplasmic HYL1 modulates miRNA-mediated translational repression

MicroRNAs (miRNAs) control various biological processes by repressing target mRNAs. In plants, miRNAs mediate target gene repression via both mRNA cleavage and translational repression. However, the mechanism underlying this translational repression is poorly understood. Here, we found that Arabidopsis thaliana HYPONASTIC LEAVES1 (HYL1), a core component of the miRNA processing machinery, regulates miRNA-mediated mRNA translation but not miRNA biogenesis when it localized in the cytoplasm. Cytoplasmic HYL1 localizes to the endoplasmic reticulum and associates with ARGONAUTE1 (AGO1) and ALTERED MERISTEM PROGRAM1. In the cytoplasm, HYL1 monitors the distribution of AGO1 onto polysomes, binds to the mRNAs of target genes, represses their translation, and partially rescues the phenotype of the hyl1 null mutant. This study uncovered another function of HYL1 and provides insight into the mechanism of plant gene regulation.

The nuclear miRNA biogenesis factor HYL1 also localizes to the cytoplasm to modulate miRNA-mediated translational repression.     

Differential metabolomic responses of PAMP-triggered immunity and effector-triggered immunity in Arabidopsis suspension cells

Introduction

The rhizobacterial tomato pathogen Pseudomonas syringae pv. tomato str. DC3000 (PstDC3000), like many plant pathogenic bacteria, can elicit hypersensitive response in non-host plant cells. PstDC3000 uses a type III protein secretion system (T3SS) to deliver effector proteins.

Objectives

We compared metabolomic responses of Arabidopsis suspension cells to a wild-type PstDC3000, a T3SS deletion mutant PstDC3000D28E, and a pathogen associated molecular pattern (PAMP) flagellin’s N-terminal domain’s 22-aa peptide (flg22) to obtain metabolomics insights into the plant cell PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI).

Methods

Using targeted HPLC-MRM-MS and untargeted GC-MS approaches, we monitored qualitative and quantitative changes of 312 metabolites in central and specialized metabolic pathways in a time-course study.

Results

The overall metabolomic changes induced by the three treatments included phenylpropanoid, flavonoid, and phytohormone biosynthetic pathways, as well as primary metabolism in amino acid and sugar biosynthesis. In addition to shared metabolites, flg22, PstDC3000D28E and PstDC3000 each caused unique metabolite changes in the course of the development of PTI and ETI.

Conclusion

PstDC3000D28E triggered PTI responses were different from those of flg22. This study has not only revealed the discernible metabolomics features associated with the flg22, PstDC3000D28E and PstDC3000 treatments, but also laid a foundation toward further understanding of metabolic regulation and responses underlying plant PTI and ETI.

     

Basidiospores of <Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis> succeed to infect barberry,while Urediniospores are blocked by non-host resistance

Stripe rust (Yellow rust) caused by Puccinia striiformis f. sp. tritici (Pst) is a major disease of wheat worldwide. The use of resistant cultivars to control Pst has been very effective, low-cost, and ecologically sound. However, virulence patterns of Pst can quickly change, which may render resistant cultivars susceptible. The discovery of infection of Berberis spp. by basidiospores of Pst in 2010 raised important concerns about the evolution of new virulent races of the pathogen. Little is known about the infection process of Berberis spp. by basidiospores of Pst and the interaction between Berberis spp. and asexual urediniospores. In this study, the interaction between Pst urediniospores and Berberis spp. was investigated at histological and cytological levels. Our results indicate that Berberis spp. expresses a continuum of layered defenses comprised of structural and chemical changes in the cell wall as well as post-haustorial hypersensitive responses to urediniospore infection. Our study also re-examines in detail the infection process of Pst basidiospores on Berberis spp. and provides useful information for further research on the molecular mechanisms governing the interaction between Berberis spp. and Pst.     

Jasmonic acid modulates xylem development by controlling polar auxin transport in vascular tissues

Development of xylem cells is affected by environmental stresses such as drought and oxidative stress, and recent findings suggested that jasmonic acid (JA) mediates this process through interaction with other phytohormones such as cytokinin. In this study, we showed that polar auxin transport regulated by PIN3 and PIN7 is involved in the JA-mediated xylem development in vascular tissues. The mutant plants that lack the activity of PIN3 and PIN7 responsible for the auxin transport developed extra xylems in vascular tissues such as the JA-treated wild-type plants. Visualization of auxin response and xylem development in the roots treated with NPA, an inhibitor of polar auxin transport, suggested that disruption of polar auxin transport is involved in the xylem phenotype of pin3 pin7 double mutants. We also found that cytokinin increases expressions of PIN3 and PIN7 responsible for the auxin transport while JA decreases only PIN7. These suggested that PIN7-mediated polar auxin transport system modulates xylem development in response to JA. The finding that JA affects auxin distribution in root vascular tissues further supported this. Collectively, these suggest that JA promotes xylem development by disrupting auxin transport in vascular tissues, and the auxin efflux genes, more especially PIN7 whose expression is suppressed by JA mediates this process.     

Defense suppression benefits herbivores that have a monopoly on their feeding site but can backfire within natural communities

Background

Plants have inducible defenses to combat attacking organisms. Hence, some herbivores have adapted to suppress these defenses. Suppression of plant defenses has been shown to benefit herbivores by boosting their growth and reproductive performance.

Results

We observed in field-grown tomatoes that spider mites (Tetranychus urticae) establish larger colonies on plants already infested with the tomato russet mite (Aculops lycopersici). Using laboratory assays, we observed that spider mites have a much higher reproductive performance on russet mite-infested plants, similar to their performance on the jasmonic acid (JA)-biosynthesis mutant def-1. Hence, we tested if russet mites suppress JA-responses thereby facilitating spider mites. We found that russet mites manipulate defenses: they induce those mediated by salicylic acid (SA) but suppress those mediated by JA which would otherwise hinder growth. This suppression of JA-defenses occurs downstream of JA-accumulation and is independent from its natural antagonist SA. In contrast, spider mites induced both JA- and SA-responses while plants infested with the two mite species together display strongly reduced JA-responses, yet a doubled SA-response. The spider mite-induced JA-response in the presence of russet mites was restored on transgenic tomatoes unable to accumulate SA (nahG), but russet mites alone still did not induce JA-responses on nahG plants. Thus, indirect facilitation of spider mites by russet mites depends on the antagonistic action of SA on JA while suppression of JA-defenses by russet mites does not. Furthermore, russet mite-induced SA-responses inhibited secondary infection by Pseudomonas syringae (Pst) while not affecting the mite itself. Finally, while facilitating spider mites, russet mites experience reduced population growth.

Conclusions

Our results show that the benefits of suppressing plant defenses may diminish within communities with natural competitors. We show that suppression of defenses via the JA-SA antagonism can be a consequence, rather than the cause, of a primary suppression event and that its overall effect is determined by the presence of competing herbivores and the distinct palette of defenses these induce. Thus, whether or not host-defense manipulation improves an herbivore’s fitness depends on interactions with other herbivores via induced-host defenses, implicating bidirectional causation of community structure of herbivores sharing a plant.

     

Further insight into the role of <Emphasis Type="Italic">KAN1</Emphasis>, a member of <Emphasis Type="Italic">KANADI</Emphasis> transcription factor family in rice

The rice EMS-derived mutant leaf adaxialized 1 (lad1) was isolated based on its upward rolling leaf phenotype. Besides the adaxially rolled leaf, many other agronomic traits were also compromised in lad1. The rolling trait was characterized by a noticeable alteration of bulliform cells in the adaxial side of the leaves. Map-based cloning showed a single nucleotide substitution in the promoter region of the KAN1 gene in lad1 mutant. Further, over-expressing and CRISPR/cas9-edited knockdown transgenic plants confirmed that KAN1 was responsible for the mutant phenotype of lad1. Yeast two-hybrid and bimolecular fluorescence complementation assay demonstrated that KAN1 can interact with the auxin response factors ARF3, ARF7 and ARF15. Physiologically, the contents of auxin (IAA), abscisic acid (ABA), jasmonic acid (JA) and gibberellin (GA) were all significantly increased in the lad1 mutant. Moreover, the GA3 content dramatically decrease in wild-type, but increased in lad1 under IAA induction. Additionally, the expression levels of several IAA and GA biosynthesis and responsive-related genes and genes involved in leaf polarity determination were altered in lad1. Therefore, we hypothesized that KAN1/ARFs protein complexes act as auxin-dependent regulatory units that play a conserved role in leaf development.     

A <Emphasis Type="Italic">pqr2</Emphasis> mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 μM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na₂WO₄, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.     

Genetic analysis and fine-mapping of a new rice mutant, <Emphasis Type="Italic">white and lesion mimic leaf1</Emphasis>

Rice (Oryza sativa L.) leaf color mutants are excellent models for studying chlorophyll biosynthesis and chloroplast development. In this study, we isolated a stable genetic white and lesion mimic leaf1 (wlml1) mutant from an ethyl methanesulfonate (EMS)-mutagenized population of the indica cultivar TN1. Compared with wild-type TN1, the wlml1 mutant had lower contents of chlorophyll and carotenoids, altered chloroplast ultrastructure, and altered regulation of genes associated with chlorophyll metabolism and chloroplast development. In addition, lesions formed on the leaves of wlml1 plants grown at 20 °C and genes related to disease resistance and antioxidant functions were up-regulated; by contrast, the mutant phenotype was partially suppressed at 28 °C. These findings indicated that WLML1 might play a role in chlorophyll metabolism and chloroplast development, as well as in biotic and abiotic stress responses. Genetic analysis showed that WLML1 was controlled by a recessive nuclear gene, and map-based cloning delimited WLML1 to a 159.7-kb region on chromosome 4 that includes 30 putative open reading frames. Based on these findings, the wlml1 mutant will be a good genetic material for further studies on chlorophyll metabolism and stress responses in rice.     

Identification and comparative analysis of <Emphasis Type="Italic">Brassica juncea</Emphasis> pathogenesis-related genes in response to hormonal,biotic and abiotic stresses

Pathogenesis-related proteins (PRs) are the antimicrobial proteins which are commonly used as signatures of defense signaling pathways and systemic acquired resistance. However, in Brassica juncea most of the PR proteins have not been fully characterized and remains largely enigmatic. In this study, full-length cDNA sequences of SA (PR1, PR2, PR5) and JA (PR3, PR12 and PR13) marker genes were isolated from B. juncea and were named as BjPR proteins. BjPR proteins showed maximum identity with known PR proteins of Brassica species. Further, expression profiling of BjPR genes were investigated after hormonal, biotic and abiotic stresses. Pre-treatment with SA and JA stimulators downregulates each other signature genes suggesting an antagonistic relationship between SA and JA in B. juncea. After abscisic acid (ABA) treatment, SA signatures were downregulated while as JA signature genes were upregulated. During Erysiphe cruciferarum infection, SA- and JA-dependent BjPR genes showed distinct expression pattern both locally and systemically, thus suggesting the activation of SA- and JA-dependent signaling pathways. Further, expression of SA marker genes decreases while as JA-responsive genes increases during drought stress. Interestingly, both SA and JA signature genes were induced after salt stress. We also found that BjPR genes displayed ABA-independent gene expression pattern during abiotic stresses thus providing the evidence of SA/JA cross talk. Further, in silico analysis of the upstream regions (1.5 kb) of both SA and JA marker genes showed important cis-regulatory elements related to biotic, abiotic and hormonal stresses.     

Disorder of trafficking system of plasma membrane and vacuole antiporter proteins causes hypersensitive response to salinity stress in <Emphasis Type="Italic">Arabidopsis Thaliana</Emphasis>

Rab GTPases play an important role in regulating intracellular vesicular trafficking in eukaryotic cells. Previously, we found that Oryza sativa rice Rab11 (OsRab11) is required for the regulation of vesicular trafficking from the trans- Golgi network (TGN) to the plasma membrane (PM) and/or vacuoles. To further elucidate the relationship between vesicular trafficking and abiotic and biotic stresses, we determined OsRab11 expression levels under several environmental stress conditions. OsRab11 expression was induced by pathogens, jasmonic acid (JA), and high salt treatment. Under high salt conditions, dominant negative OsRab11(S28N) mutant plants exhibited a hypersensitive phenotype similar to that of sos1-1, whereas overexpressed-OsRab11 plants showed resistance to high salt stress. When the expression of vacuolar and PM Na⁺/H⁺ antiporter genes such as AtNHX1, AtNHX2, and AtSOS1 was examined, there was no significant difference between the wild-type and OsRab11(S28N) mutant plants. However, PM trafficking of AtSOS1-green fluorescent protein (GFP) in 35S::AtSOS1-GFP sos1-1 plants was severely impaired by T7-OsRab11(S28N) expression. Similarly, vacuolar trafficking of AtNHX2-GFP was inhibited by T7-OsRab11 (S28N) expression. These results indicate that trafficking of PM and vacuolar antiporter proteins by OsRab11 is important for high salt stress resistance.