Sweet revenge: AtSWEET12 in plant defense against bacterial pathogens by apoplastic sucrose limitation

Depriving bacterial pathogens of sugars is a potential plant defense strategy. The relevance of SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS (SWEETs) in plant susceptibility to pathogens has been established, but their role in plant defense remains unknown. We identified Arabidopsis thaliana SWEETs (AtSWEETs) involved in defense against nonhost and host Pseudomonas syringae pathogens through reverse genetic screening of atsweet1-17 mutants. Double/triple mutant, complementation, and overexpression line analysis, and apoplastic sucrose estimation studies revealed that AtSWEET12 suppresses pathogen multiplication by limiting sucrose availability in the apoplast. Localization studies suggested that plant defense occurred via increased plasma membrane targeting of AtSWEET12 with concomitant AtSWEET11 protein reduction. Moreover, the heterooligomerization of AtSWEET11 and AtSWEET12 was involved in regulating sucrose transport. Our results highlight a PAMP-mediated defense strategy against foliar bacterial pathogens whereby plants control AtSWEET11-mediated sucrose efflux in the apoplast through AtSWEET12. We uncover a fascinating new mechanism of pathogen starvation as a broad-spectrum disease resistance mechanism in parallel with existing immune pathways.

The Natural Antimicrobial trans-Cinnamaldehyde Interferes with UDP-N-Acetylglucosamine Biosynthesis and Cell Wall Homeostasis in Listeria monocytogenes

Trans-cinnamaldehyde (t-CIN), an antimicrobial compound from cinnamon essential oil, is of interest because it inhibits various foodborne pathogens. In the present work, we investigated the antimicrobial mechanisms of t-CIN in Listeria monocytogenes using a previously isolated yvcK::Himar1 transposon mutant which shows hypersensitivity to t-CIN. Time-lapse microscopy revealed that t-CIN induces a bulging cell shape followed by lysis in the mutant. Complementation with wild-type yvcK gene completely restored the tolerance of yvcK::Himar1 strain to t-CIN and the cell morphology. Suppressor mutants which partially reversed the t-CIN sensitivity of the yvcK::Himar1 mutant were isolated from evolutionary experiments. Three out of five suppression mutations were in the glmU-prs operon and in nagR, which are linked to the biosynthesis of the peptidoglycan precursor uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc). GlmU catalyzes the last two steps of UDP-GlcNAc biosynthesis and NagR represses the uptake and utilization of N-acetylglucosamine. Feeding N-acetylglucosamine or increasing the production of UDP-GlcNAc synthetic enzymes fully or partially restored the t-CIN tolerance of the yvcK mutant. Together, these results suggest that YvcK plays a pivotal role in diverting substrates to UDP-GlcNAc biosynthesis in L. monocytogenes and that t-CIN interferes with this pathway, leading to a peptidoglycan synthesis defect.

Divergence of three BRX homoeologs in Brassica rapa and its effect on leaf morphology

The leafy head characteristic is a special phenotype of Chinese cabbage resulting from artificial selection during domestication and breeding. BREVIS RADIX (BRX) has been suggested to control root elongation, shoot growth, and tiller angle in Arabidopsis and rice. In Brassica rapa, three BrBRX homoeologs have been identified, but only BrBRX.1 and BrBRX.2 were found to be under selection in leaf-heading accessions, indicating their functional diversification in leafy head formation. Here, we show that these three BrBRX genes belong to a plant-specific BRX gene family but that they have significantly diverged from other BRX-like members on the basis of different phylogenetic classifications, motif compositions and expression patterns. Moreover, although the expression of these three BrBRX genes differed, compared with BrBRX.3, BrBRX.1, and BrBRX.2 displayed similar expression patterns. Arabidopsis mutant complementation studies showed that only BrBRX.1 could rescue the brx root phenotype, whereas BrBRX.2 and BrBRX.3 could not. However, overexpression of each of the three BrBRX genes in Arabidopsis resulted in similar pleiotropic leaf phenotypes, including epinastic leaf morphology, with an increase in leaf number and leaf petiole length and a reduction in leaf angle. These leaf traits are associated with leafy head formation. Further testing of a SNP (T/C) in BrBRX.2 confirmed that this allele in the heading accessions was strongly associated with the leaf-heading trait of B. rapa. Our results revealed that all three BrBRX genes may be involved in the leaf-heading trait, but they may have functionally diverged on the basis of their differential expression.

Highly pleiotropic functions of CYP78As and AMP1 are regulated in non-cell-autonomous/organ-specific manners

The cytochrome P450 CYP78A5/KLUH in Arabidopsis thaliana is predicted to be involved in the synthesis of a mobile signal molecule that has a pleiotropic function that is distinct from classical phytohormones. CYP78A5 has five close relatives in Arabidopsis. We first investigated their functions, focusing on the plastochron, leaf size, and leaf senescence. Our analyses revealed that CYP78A5 and CYP78A7 are involved in the plastochron and leaf size, and CYP78A6 and CYP78A9 are involved in leaf senescence. Complementation analyses using heterologous promoters and expression analyses suggested that CYP78A isoforms have a common biochemical function and are functionally differentiated via organ-specific expression. The altered meristem program1 (amp1) carboxypeptidase mutant shows a phenotype very similar to that of the cyp78a5 mutant. Complementation analyses using boundary- and organizing center-specific promoters suggested that both CYP78A5 and AMP1 act in a non-cell-autonomous manner. Analyses of multiple cyp78a mutants and crosses between cyp78a and amp1 mutants revealed that AMP1/LIKE AMP1 (LAMP1) and CYP78A isoforms regulate plastochron length and leaf senescence in the same genetic pathway, whereas leaf size is independently regulated. Furthermore, we detected feedback regulation between CYP78A6/CYP78A9 and AMP1 at the gene expression level. These observations raise the possibility that AMP1 and CYP78A isoforms are involved in the synthesis of the same mobile signal molecule, and suggest that AMP1 and CYP78A signaling pathways have a very close, albeit complex, functional relationship.s

The Pentatricopeptide Repeat Protein MEF100 Is Required for the Editing of Four Mitochondrial Editing Sites in Arabidopsis

In Arabidopsis thaliana there are more than 600 C-to-U RNA editing events in the mitochondria and at least 44 in the chloroplasts. Pentatricopeptide repeat (PPR) proteins provide the specificity for these reactions. They recognize RNA sequences in a partially predictable fashion via key amino acids at the fifth and last position in each PPR motif that bind to individual ribonucleotides. A combined approach of RNA-Seq, mutant complementation, electrophoresis of mitochondrial protein complexes and Western blotting allowed us to show that MEF100, a PPR protein identified in a genetic screen for mutants resistant to an inhibitor of γ -glutamylcysteine synthetase, is required for the editing of nad1-493, nad4-403, nad7-698 and ccmFN2-356 sites in Arabidopsis mitochondria. The absence of editing in mef100 leads to a decrease in mitochondrial Complex I activity, which probably explains the physiological phenotype. Some plants have lost the requirement for MEF100 at one or more of these sites through mutations in the mitochondrial genome. We show that loss of the requirement for MEF100 editing leads to divergence in the MEF100 binding site.

A recombineering pipeline to clone large and complex genes in Chlamydomonas

The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is PCR or DNA synthesis dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kilobases. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.

Organization and control of the ascorbate biosynthesis pathway in plants

ABSTRACTThe enzymatic steps involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, and l-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP and l-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complemented vtc2 null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes in N. benthamiana supported the role of VTC2 as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 and l-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-l-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.

A recombineering pipeline to clone large and complex genes in Chlamydomonas

The ability to clone genes has driven fundamental advances in cell and molecular biology, enabling researchers to introduce precise mutations, generate fluorescent protein fusions for localization and to confirm genetic causation by mutant complementation. Most gene cloning is PCR or DNA synthesis dependent, which can become costly and technically challenging as genes increase in size and particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, with a high percentage of genes containing complex sequence structures, an average genomic GC content of 64% and gene expression requiring regular introns for stable transcription. Here we overcome these challenges via the development of a recombineering pipeline that enables the rapid parallel cloning of genes from a Chlamydomonas BAC collection. We show the method can successfully retrieve large and complex genes that PCR-based methods have previously failed to clone, including genes as large as 23 kilobases, thus making previously technically challenging genes to study now amenable to cloning. We initially applied the pipeline to 12 targets with a 92% cloning success rate. We then developed a high-throughput approach and targeted 191 genes relating to the Chlamydomonas CO2 concentrating mechanism (CCM) with an overall cloning success rate of 77% that is independent of gene size. Localization of a subset of CCM targets has confirmed previous mass spectrometry data and identified new pyrenoid components. To expand the functionality of our system, we developed a series of localization vectors that enable complementation of Chlamydomonas Library Project mutants and enable protein tagging with a range of fluorophores. Vectors and detailed protocols are available to facilitate the easy adoption of this method by the Chlamydomonas research community. We envision that this technology will open up new possibilities in algal and plant research and be complementary to the Chlamydomonas mutant library.

Melon ethylene-mediated transcriptome and methylome dynamics provide insights to volatile production

During climacteric ripening large-scale transcriptional modifications are governed by ethylene. While ripening-related chromatin modifications are also known to occur, a direct connection between these factors has not been demonstrated. We characterized ethylene-mediated transcriptome modification, genome methylation dynamics, and their relation to organoleptic modifications during fruit ripening in the climacteric melon and an ethylene repressed line where the fruit-specific ACC oxidase 1 (ACO1) gene was targeted by antisense. The ACO1 antisense line exhibited mainly reduced transcriptional repression of ripening-related genes associated with DNA CHH hypomethylation at the onset of ripening. Additionally, transcription of a small set of ethylene-induced genes, including known ripening-associated genes, was inhibited by ACO1 repression and this inhibition was associated with CG hypermethylation. In the ACO1 antisense line, the accumulation of aromatic compounds, which are mainly derived from the catabolism of amino acids, is known to be inhibited. One of the ethylene-mediated transcriptionally up-regulated genes, CmTHA1, encoding a threonine aldolase, exhibited differential cytosine methylation. Threonine aldolase catalyzes the conversion of L-threonine/L-allo threonine to glycine and acetaldehyde and thus is likely involved in threonine-dependent ethyl ester biosynthesis. Yeast mutant complementation and incubation of melon discs with labeled threonine verified CmTHA1 threonine aldolase activity, revealing an additional ethylene-dependent amino acid catabolism branch involved in climacteric melon ripening.

Functional Gene Network of Prenyltransferases in Arabidopsis thaliana

Prenyltransferases (PTs) are enzymes that catalyze prenyl chain elongation. Some are highly similar to each other at the amino acid level. Therefore, it is difficult to assign their function based solely on their sequence homology to functional orthologs. Other experiments, such as in vitro enzymatic assay, mutant analysis, and mutant complementation are necessary to assign their precise function. Moreover, subcellular localization can also influence the functionality of the enzymes within the pathway network, because different isoprenoid end products are synthesized in the cytosol, mitochondria, or plastids from prenyl diphosphate (prenyl-PP) substrates. In addition to in vivo functional experiments, in silico approaches, such as co-expression analysis, can provide information about the topology of PTs within the isoprenoid pathway network. There has been huge progress in the last few years in the characterization of individual Arabidopsis PTs, resulting in better understanding of their function and their topology within the isoprenoid pathway. Here, we summarize these findings and present the updated topological model of PTs in the Arabidopsis thaliana isoprenoid pathway.