Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity

Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N⁶-(∆²-isopentenyl)adenine and dimethylated N⁶-(∆²-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N⁶-(∆²-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N⁶-(∆²-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.The balance of phytohormones, such as cytokinins (CKs) and auxins, is finely controlled to maintain proper plant growth and development but is often disturbed following pathogen infection (Robert-Seilaniantz et al., 2007; Pieterse et al., 2012). As a virulence strategy, many phytopathogens synthesize phytohormones that cause aberrant organogenesis and modulate primary carbon metabolism that ultimately aids disease establishment (Jameson, 2000; Robert-Seilaniantz et al., 2007). For several pathogens, CK production is essential for virulence, and they carry genes for CK biosynthesis in a harbored plasmid (Jameson, 2000). Fungal pathogens employ CKs to form green islands with delayed senescence, whereas bacterial pathogens develop gall structures (Sakakibara et al., 2005; Walters et al., 2008; Giron et al., 2013). Rhodococcus fascians is a gram-positive actinomycete that causes symptoms ranging from leaf deformation to differentiated shooty outgrowths known as leafy galls in more than 150 different plant species (Goethals et al., 2001; Stes et al., 2011). In ornamental plants, such infections reduce their value and contribute to economic losses worldwide (Putnam and Miller, 2007). Leafy gall symptoms are reminiscent of CK overproduction and can be partially induced by exogenous application of CKs (Thimann and Sachs, 1966; Eason et al., 1996). Although several CKs have been isolated from R. fascians culture filtrates, a clear correlation with pathogenesis is lacking partially owing to the low concentration of bacterial CKs (Eason et al., 1996). A synergistic action by a mixture of bacterially produced CKs has been proposed, leading to persistent accumulation of CKs locally (Pertry et al., 2009). Nevertheless, to date, no virulence-associated CK analogs have been identified that could contribute to the infection symptoms.Naturally occurring CKs are adenine derivatives with different side chains at the N6 position. Major plant CKs are N⁶-prenylated adenine derivatives such as N⁶-(Δ²-isopentenyl)adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin, collectively known as isoprenoid CKs (Sakakibara, 2006). Among them, iP and tZ are the major CKs in Arabidopsis (Arabidopsis thaliana). iP is synthesized by adenosine phosphate-isopentenyl transferase (IPT) using dimethylallyl diphosphate (DMAPP) and adenosine phosphate as substrates (Sakakibara, 2006). tZ is formed by hydroxylation of the trans-end of the prenyl side chain of the iP nucleotide. CK homeostasis is governed by both biosynthesis and catabolism and has an important regulatory role in plant growth (Sakakibara, 2006; Werner et al., 2006). CK oxidase/dehydrogenase (CKX) is responsible for an irreversible reaction cleaving the unsaturated isoprenoid side chain that results in the formation of adenine and the corresponding aldehyde (Werner et al., 2006). In Arabidopsis, CKs are perceived by a subset of sensory His kinases, ARABIDOPSIS HIS KINASE2 (AHK2) to AHK4, which undergo a His-Asp phosphorelay leading to induction of direct target genes including type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes (Kieber and Schaller, 2010). This two-component signaling system has been implicated in mediating basal and pathogen-induced plant immunity (Choi et al., 2010; Argueso et al., 2012). For instance, infection of Arabidopsis plants by R. fascians reportedly activates type-A ARR5 expression with increased expression of AHK3 and AHK4, resulting in mitotic cell divisions that arrest the infected leaves in a meristematic state to establish a nutrient-rich niche (Depuydt et al., 2008, 2009; Pertry et al., 2010; Stes et al., 2011). As the infection progresses, IPT genes are switched off, whereas the expression of all CKX genes are strongly induced in symptomatic tissues (Depuydt et al., 2008).The virulence determinant of R. fascians is located within the fasciation (fas) locus, an operon encoding several genes involved in CK metabolism, indicating that CKs are essential for this plant-pathogen interaction (Stes et al., 2011). fas4 encodes IPT that catalyzes the rate-limiting step of CK biosynthesis and is vital for virulence (Stes et al., 2013). Interestingly, two methyltransferase-like genes are present upstream of the fas gene, whose functions have been unknown. Despite the presence of the fas genes in R. fascians, fewer known CKs have been detected compared with other gall-causing pathogens such as Pantoea agglomerans, Agrobacterium tumefaciens, and Pseudomonas savastanoi (Goethals et al., 2001). Further, the leafy gall phenotype is unique, not invoked by any of the above-mentioned pathogens, implying that the virulence of R. fascians might not be due to typical CKs alone (Goethals et al., 2001). R. fascians has long been hypothesized to produce CK analogs using similar or modified substrates (Goethals et al., 2001; Galis et al., 2005; Stes et al., 2011), but no such molecules have been discovered so far. Here, we report the identification and mode of biosynthesis for methylated cytokinins (MeCKs) as hormone mimics from R. fascians. These compounds are synthesized by two methyltransferases and FAS4. Their CK-like activity and higher in planta stability suggest a role for the methylated analogs as CK mimics that foster efficient pathogenesis.     

Phylogeny and chromosomal variations in East Asian Carex, Siderostictae group (Cyperaceae), based on DNA sequences and cytological data

Carex (Cyperaceae) is one of the largest genera of the flowering plants, and comprises more than 2,000 species. In Carex, section Siderostictae with broader leaves distributed in East Asia is thought to be an ancestral group. We aimed to clarify the phylogenetic relationships and chromosomal variations within the section Siderostictae, and to examine the relationship of broad-leaved species of the sections Hemiscaposae and Surculosae from East Asia, inferred from DNA sequences and cytological data. Our results indicate that a monophyletic Siderostictae clade, including the sections Hemiscaposae, Siderostictae and Surculosae, as the earliest diverging group in the tribe Cariceae. Low chromosome numbers, 2n = 12 or 24, with large sizes were observed in these three sections. Our results suggest that the genus Carex might have originated or relictly restricted in the East Asia. Geographical distributions of diploid species are restricted in narrower areas, while those of tetraploid species are wider in East Asia. It is concluded that chromosomal variations in Siderostictae clade may have been caused by polyploidization and that tetraploid species may have been able to exploit their habitats by polyploidization.     

The effects of the photoperiod-insensitive alleles,se13, hd1 and ghd7, on yield components in rice

Flowering time is closely associated with grain yield in rice (Oryza sativa L.). In temperate regions, seasonal changes in day length (known as the photoperiod) are an important environmental cue for floral initiation. The timing of flowering is important not only for successful reproduction, but also for determining the ideal balance between vegetative growth and reproductive growth duration. Recent molecular genetics studies have revealed key flowering time genes responsible for photoperiod sensitivity. In this study, we investigated the effect of three recessive photoperiod-insensitive alleles, se13, hd1 and ghd7, on yield components in rice under Ehd1-deficient genetic background conditions to ensure vegetative growth of each line. We found that se13-bearing plants had fewer panicles, hd1-bearing plants showed decreased grain-filling percentage, and ghd7-bearing plants appeared to have fewer grains per panicle and fewer secondary branches. Our results indicate that the pleiotropic effects of photoperiod-insensitive genes on yield components are independent of short vegetative growth. This will provide critical information which can be used to create photoperiod-insensitive varieties that can be adapted to a wide range of latitudes.     

Complex I generated, mitochondrial matrix-directed superoxide is released from the mitochondria through voltage dependent anion channels

Mitochondrial complex I has previously been shown to release superoxide exclusively towards the mitochondrial matrix, whereas complex III releases superoxide to both the matrix and the cytosol. Superoxide produced at complex III has been shown to exit the mitochondria through voltage dependent anion channels (VDAC). To test whether complex I-derived, mitochondrial matrix-directed superoxide can be released to the cytosol, we measured superoxide generation in mitochondria isolated from wild type and from mice genetically altered to be deficient in MnSOD activity (TnIFastCreSod2(fl/fl)). Under experimental conditions that produce superoxide primarily by complex I (glutamate/malate plus rotenone, GM+R), MnSOD-deficient mitochondria release ～4-fold more superoxide than mitochondria isolated from wild type mice. Exogenous CuZnSOD completely abolished the EPR-derived GM+R signal in mitochondria isolated from both genotypes, evidence that confirms mitochondrial superoxide release. Addition of the VDAC inhibitor DIDS significantly reduced mitochondrial superoxide release (～75%) in mitochondria from either genotype respiring on GM+R. Conversely, inhibition of potential inner membrane sites of superoxide exit, including the matrix face of the mitochondrial permeability transition pore and the inner membrane anion channel did not reduce mitochondrial superoxide release in the presence of GM+R in mitochondria isolated from either genotype. These data support the concept that complex I-derived mitochondrial superoxide release does indeed occur and that the majority of this release occurs through VDACs.     

Macrophage-capping protein as a tissue biomarker for prediction of response to gemcitabine treatment and prognosis in cholangiocarcinoma

Cholangiocarcinoma is one of the deadliest malignancies worldwide. Recent studies reported that treatment with gemcitabine was effective in prolonging survival. However, as the treatment only benefited a limited subset of patients, selection of patients before treatment is required. To discover biomarkers predictive of the response to gemcitabine treatment in cholangiocarcinoma, we examined the proteome of three types of material resource; ten cell lines, nine xenografts and nine surgically resected primary tumors from patients who exhibited different response to gemcitabine treatment. Two-dimensional difference gel electrophoresis generated quantitative protein expression profiles including 3571 protein spots. We detected 172 protein spots with significant correlation with response to gemcitabine treatment. All proteins corresponding to these 172 protein spots were identified by mass spectrometry. We found that the macrophage-capping protein (CapG) was associated with response to gemcitabin treatment in all three types of material source. Immunohistochemical validation in an additional set of 196 cholangiocarcinoma cases revealed that CapG expression was associated with lymphatic invasion status and overall survival. Multivariate analysis showed that CapG protein expression was an independent prognostic factor for overall survival. In conclusion, CapG was identified as a novel candidate biomarker to predict response to gemcitabine treatment and survival in cholangiocarcinoma.     

Transformation of Jatropha curcas L. for production of larger seeds and increased amount of biodiesel

The development of green energy is important to mitigate global warming. Jatropha (Jatropha curcas L.) is a promising candidate for the production of alternative biofuel, which could reduce the burden on the Earth’s resources. Jatropha seeds contain a large quantity of lipids that can be used to produce biofuel, and the rest of the plant has many other uses. Currently, techniques for plant genetic transformation are extensively employed to study, create, and improve the specific characteristics of the target plant. Successful transformation involves the alteration of plants and their genetic materials. The aim of this study was to generate Jatropha plants that can support biofuel production by increasing their seed size using genes found via the rice FOX-hunting system. The present study improved previous protocols, enabling the production of transgenic Jatropha in two steps: the first step involved using auxins and dark incubation to promote root formation in excised shoots and the second step involved delaying the timing of antibiotic selection in the cultivation medium. Transgenic plants were subjected to PCR analysis; the transferred gene expression was confirmed via RT-PCR and the ploidy level was investigated. The results suggest that the genes associated with larger seed size in Arabidopsis thaliana, which were found using the rice FOX-hunting system, produce larger seeds in Jatropha.     

Loss of NECROTIC SPOTTED LESIONS 1 associates with cell death and defense responses in Arabidopsis thaliana

We isolated a lesion mimic mutant, n ecrotic s potted l esions 1 (nsl1), from Ds-tagged Arabidopsis thaliana accession No-0. The nsl1 mutant exhibits a growth retardation phenotype and develops spotted necrotic lesions on its rosette and cauline leaves. These phenotypes occur in the absence of pathogens indicating that nsl1 mutants may constitutively express defense responses. Consistent with this idea, nsl1 accumulates high levels of callose and autofluorescent phenolic compounds localized to the necrotic lesions. Furthermore RNA gel blot analysis revealed that genes associated with disease resistance activation are upregulated in the nsl1 mutants and these plants contain elevated levels of salicylic acid (SA). Crossing nsl1 with an SA deficient mutant, eds16-1, revealed that the nsl1 lesions and growth retardation are dependent upon SA. The nsl1 phenotypes are not suppressed under either the rar1-10 or sgt1b-1 genetic background. NSL1 encodes a novel 612aa protein which contains a membrane-attack complex/perforin (MACPF) domain, which is conserved in bacteria, fungi, mammals and plants. The possible modes of action of NSL1 protein in negative regulation of cell death programs and defense responses are discussed.