The Arabidopsis Rho of Plants GTPase AtROP6 Functions in Developmental and Pathogen Response Pathways

How plants coordinate developmental processes and environmental stress responses is a pressing question. Here, we show that Arabidopsis (Arabidopsis thaliana) Rho of Plants6 (AtROP6) integrates developmental and pathogen response signaling. AtROP6 expression is induced by auxin and detected in the root meristem, lateral root initials, and leaf hydathodes. Plants expressing a dominant negative AtROP6 (rop6^DN) under the regulation of its endogenous promoter are small and have multiple inflorescence stems, twisted leaves, deformed leaf epidermis pavement cells, and differentially organized cytoskeleton. Microarray analyses of rop6^DN plants revealed that major changes in gene expression are associated with constitutive salicylic acid (SA)-mediated defense responses. In agreement, their free and total SA levels resembled those of wild-type plants inoculated with a virulent powdery mildew pathogen. The constitutive SA-associated response in rop6^DN was suppressed in mutant backgrounds defective in SA signaling (nonexpresser of PR genes1 [npr1]) or biosynthesis (salicylic acid induction deficient2 [sid2]). However, the rop6^DN npr1 and rop6^DN sid2 double mutants retained the aberrant developmental phenotypes, indicating that the constitutive SA response can be uncoupled from ROP function(s) in development. rop6^DN plants exhibited enhanced preinvasive defense responses to a host-adapted virulent powdery mildew fungus but were impaired in preinvasive defenses upon inoculation with a nonadapted powdery mildew. The host-adapted powdery mildew had a reduced reproductive fitness on rop6^DN plants, which was retained in mutant backgrounds defective in SA biosynthesis or signaling. Our findings indicate that both the morphological aberrations and altered sensitivity to powdery mildews of rop6^DN plants result from perturbations that are independent from the SA-associated response. These perturbations uncouple SA-dependent defense signaling from disease resistance execution.Rho of Plants (ROPs), also known as RACs (for clarity, the ROP nomenclature will be used throughout this article), comprise a plant-specific group of Rho family small G proteins. Like other members of the Ras superfamily of small G proteins, ROPs function as molecular switches, existing in a GTP-bound “on” state and a GDP-bound “off” state. In the GTP-bound state, ROPs interact with specific effectors that transduce downstream signaling or function as scaffolds for interaction with additional effector molecules (Berken and Wittinghofer, 2008). Conserved point mutations in the G1 (P loop) Gly-15 or the G3 (switch II) Gln-64, which abolish GTP hydrolysis, or the G1 Thr-20 or G4 Asp-121 that compromise GDP/GTP exchange, can form either constitutively active or dominant negative mutants, respectively (Feig, 1999; Berken et al., 2005; Berken and Wittinghofer, 2008; Sorek et al., 2010). Primarily based on studies with neomorphic mutants, ROPs have been implicated in the regulation of cytoskeleton organization and dynamics, vesicle trafficking, auxin transport and response, abscisic acid (ABA) response, and response to pathogens (Nibau et al., 2006; Yalovsky et al., 2008; Yang, 2008; Lorek et al., 2010; Wu et al., 2011; and refs. therein).In Arabidopsis (Arabidopsis thaliana), there are 11 ROP proteins (Winge et al., 1997). Assigning specific functions to individual members of this family is difficult, however, because ROPs are functionally redundant. A ROP10 loss-of-function mutant was reported to be ABA hypersensitive (Zheng et al., 2002), displaying enhanced expression of tens of genes in response to ABA treatments (Xin et al., 2005). However, in the absence of exogenous ABA, gene expression in the rop10 mutant was similar to that in wild-type plants (Xin et al., 2005). Loss of leaf epidermis pavement cell polarity was reported for rop4 rop2-RNAi (for RNA interference) double mutant plants (Fu et al., 2005). Mild changes in pavement and hypocotyl cell structure and microtubule (MT) organization were reported for a rop6 loss-of-function mutant (Fu et al., 2009).The involvement of ROPs in auxin-regulated development has been addressed in several studies (Wu et al., 2011). Ectopic expression of a dominant negative ROP2 (rop2^DN) mutant under regulation of the 35S promoter resulted in a loss of apical dominance and a reduction in the number of lateral roots. In contrast, ectopic expression of constitutively active ROP2 (rop2^CA) caused an increase in the number of lateral roots and an enhanced decrease in primary root length in response to auxin. Consistent with these findings, the expression of a constitutively active NtRAC1 in tobacco (Nicotiana tabacum) protoplasts induced the expression of auxin-regulated genes in the absence of auxin and promoted the formation of protein nuclear bodies containing components of the proteasome and COP9 signalosome (Tao et al., 2002, 2005; Wu et al., 2011). The ROP effector ICR1 (for interactor of constitutively active ROP1) regulates polarized secretion and is required for polar auxin transport (Lavy et al., 2007; Bloch et al., 2008; Hazak et al., 2010; Hazak and Yalovsky, 2010). In the root, local auxin gradients induce the accumulation of ROPs in trichoblasts at the site of future root hair formation (Fischer et al., 2006). Recently, it was shown that interdigitation of leaf epidermis pavement cells depends on Auxin-Binding Protein1 (ABP1)-mediated ROP activation (Xu et al., 2010). Taken together, these data indicate that ROPs are involved in both mediating the auxin response and facilitating directional auxin transport. It is still unclear, however, which ROPs function in these processes.ROP function was linked to plant defense responses in several studies. In rice (Oryza sativa), OsRAC1 is a positive regulator of the hypersensitive response, possibly through interactions with the NADPH oxidase RbohB, Required for Mla12 Resistance, and Heat Shock Protein90 (Ono et al., 2001; Thao et al., 2007; Wong et al., 2007). Interestingly, other members of the rice ROP family, namely RAC4 and RAC5, are negative regulators of resistance to the rice blast pathogen Magnaporthe grisea (Chen et al., 2010). Similar to rice, when expressed in tobacco, dominant negative OsRAC1 suppressed the hypersensitive response (Moeder et al., 2005). In barley (Hordeum vulgare), several constitutively active ROP/RAC mutants and a MT-associated ROPGAP1 loss-of-function mutant enhanced susceptibility to the powdery mildew Blumeria graminis f. sp. hordei (Bgh). The activated ROP-enhanced susceptibility to Bgh was attributed to disorganization of the actin cytoskeleton and was shown to depend on Mildew Resistance Locus O (MLO; Schultheiss et al., 2002, 2003; Opalski et al., 2005; Hoefle et al., 2011). In barley, three ROP proteins, HvRACB, HvRAC1, and HvRAC3, were linked to both development and pathogen response (Schultheiss et al., 2005; Pathuri et al., 2008; Hoefle et al., 2011).We have analyzed the function of the Arabidopsis AtROP6 (ROP6) by characterizing its expression pattern and its regulation by auxin and the phenotype of plants that express rop6^DN under the regulation of its endogenous promoter. The utilization of the dominant negative mutant overcame functional redundancy, while expression under the regulation of the endogenous promoter enabled the analysis of ROP6 function in a developmental context. Phenotypic and gene expression analyses indicate that ROP6 functions in developmental, salicylic acid (SA)-dependent, and SA-independent defense response pathways.     

Specification of Arabidopsis floral meristem identity by repression of flowering time genes

Flowering plants produce floral meristems in response to intrinsic and extrinsic flowering inductive signals. In Arabidopsis, the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) are activated to play a pivotal role in specifying floral meristems during floral transition. We show here that the emerging floral meristems require AP1 to partly specify their floral identities by directly repressing a group of flowering time genes, including SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1). In wild-type plants, these flowering time genes are normally downregulated in emerging floral meristems. In the absence of AP1, these genes are ectopically expressed, transforming floral meristems into shoot meristems. By post-translational activation of an AP1-GR fusion protein and chromatin immunoprecipitation assays, we further demonstrate the repression of these flowering time genes by induced AP1 activity and in vivo AP1 binding to the cis-regulatory regions of these genes. These findings indicate that once AP1 is activated during the floral transition, it acts partly as a master repressor in floral meristems by directly suppressing the expression of flowering time genes, thus preventing the continuation of the shoot developmental program.     

Oncofetal H19 RNA promotes tumor metastasis

The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-β and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-β treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.     

Bulb and Root Rot in Lily (Lilium longiflorum) and Onion (Allium cepa) in Israel

In the past 10 years, there has been a substantial increase in reports, from growers and extension personnel, on bulb and root rots in lily (Lilium longiflorum) in Israel. Rot in these plants, when grown as cut flowers, caused serious economic damage expressed in reduction in yield and quality. In lily, the fungal pathogens involved in the rot were characterized as binucleate Rhizoctonia AG‐A, Rhizoctonia solani, Pythium oligandrum, Fusarium proliferatum (white and purple isolates) and F. oxysporum, using morphological and molecular criteria. These fungi were the prevalent pathogens in diseased plants collected from commercial greenhouses. Pathogenicity trials were conducted on lily bulbs and onion seedlings under controlled conditions in a greenhouse to complete Koch's postulates. Disease symptoms on lily were most severe in treatments inoculated with binucleate Rhizoctonia AG‐A, P. oligandrum and F. proliferatum. Plant height was lower in the above treatments compared with the control plants. The least aggressive fungus was R. solani. In artificial inoculations of onion, seedling survival was significantly affected by all fungi. The most pathogenic fungus was F. proliferatum w and the least were isolates of F. oxysporum (II and III). All fungi were successfully re‐isolated from the inoculated plants.     

Alopecia, neurological defects, and endocrinopathy syndrome caused by decreased expression of RBM28, a nucleolar protein associated with ribosome biogenesis

Single-gene disorders offer unique opportunities to shed light upon fundamental physiological processes in humans. We investigated an autosomal-recessive phenotype characterized by alopecia, progressive neurological defects, and endocrinopathy (ANE syndrome). By using homozygosity mapping and candidate-gene analysis, we identified a loss-of-function mutation in RBM28, encoding a nucleolar protein. RBM28 yeast ortholog, Nop4p, was previously found to regulate ribosome biogenesis. Accordingly, electron microscopy revealed marked ribosome depletion and structural abnormalities of the rough endoplasmic reticulum in patient cells, ascribing ANE syndrome to the restricted group of inherited disorders associated with ribosomal dysfunction.     

Filamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration

Caveolin-1 is an essential structural constituent of caveolae which is involved in regulation of mitogenic signaling and oncogenesis. Caveolin-1 has been implicated in cell migration but its exact role and mechanism of action in this process remained obscure. We have previously reported that expression of caveolin-1 in stably transfected MCF-7 human breast cancer (MCF-7/Cav1) cells up-regulates phosphorylation of a putative Akt substrate protein, designated pp340 [D. Ravid, S. Maor, H. Werner, M. Liscovitch, Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling, Oncogene 24 (2005) 1338-1347.]. We now show, using differential detergent extraction, SDS-PAGE and mass spectrometry, that the major protein in the pp340 band is the actin filament cross-linking protein filamin A. The identity of pp340 as filamin A was confirmed by immunoprecipitation of pp340 with specific filamin A antibodies. RT-PCR, flow cytometry and Western blot analyses show that filamin A mRNA and protein levels are respectively 3.5- and 2.5-fold higher in MCF-7/Cav1 cells than in MCF-7 cells. Basal filamin A phosphorylation on Ser-2152, normalized to total filamin A levels, is 7.8-fold higher in MCF-7/Cav1 than in MCF-7 cells. Insulin-like growth factor-I (IGF-I) stimulates phosphorylation of filamin A on Ser-2152 in MCF-7 cells and further enhances Ser-2152 phosphorylation over its already high basal level in MCF-7/Cav1 cells. The effect of IGF-I is inhibited by the PI3K inhibitor wortmannin, indicating that IGF-I-stimulated phosphorylation of filamin A occurs via the PI3K/Akt pathway. Co-immunoprecipitation experiments have confirmed a previous report showing that filamin A and caveolin-1 co-exist in a complex and have revealed the presence of active phospho-Akt in this complex. Ser-2152 phosphorylation of filamin A has been implicated in cancer cell migration. Accordingly, caveolin-1 expression dramatically enhances IGF-I-dependent MCF-7 cell migration. These data indicate that caveolin-1 specifies filamin A as a novel target for Akt-mediated filamin A Ser-2152 phosphorylation thus mediating the effects of caveolin-1 on IGF-I-induced cancer cell migration.     

Chirality-based signatures of local protein environments in two-dimensional optical spectroscopy of two species photosynthetic complexes of green sulfur bacteria: simulation study

Two-dimensional electronic chirality-induced signals of excitons in the photosynthetic Fenna-Matthews-Olson complex from two species of green sulfur bacteria (Chlorobium tepidum and Prosthecochloris aestuarii) are compared. The spectra are predicted to provide sensitive probes of local protein environment of the constituent bacteriochlorophyll a chromophores and reflect electronic structure variations (site energies and couplings) of the two complexes. Pulse polarization configurations are designed that can separate the coherent and incoherent exciton dynamics contributions to the two-dimensional spectra.