Redirection of auxin flow in Arabidopsis thaliana roots after infection by root-knot nematodes

Plant auxin efflux and influx proteins redirect the plant hormone auxin towards the feeding site upon root-knot nematode infection in Arabidopsis thaliana roots.Plant-parasitic root-knot nematodes induce the formation of giant cells within the plant root, and it has been recognized that auxin accumulates in these feeding sites. Here, we studied the role of the auxin transport system governed by AUX1/LAX3 influx proteins and different PIN efflux proteins during feeding site development in Arabidopsis thaliana roots. Data generated via promoter-reporter line and protein localization analyses evoke a model in which auxin is being imported at the basipetal side of the feeding site by the concerted action of the influx proteins AUX1 and LAX3, and the efflux protein PIN3. Mutants in auxin influx proteins AUX1 and LAX3 bear significantly fewer and smaller galls, revealing that auxin import into the feeding sites is needed for their development and expansion. The feeding site development in auxin export (PIN) mutants was only slightly hampered. Expression of some PINs appears to be suppressed in galls, probably to prevent auxin drainage. Nevertheless, a functional PIN4 gene seems to be a prerequisite for proper nematode development and gall expansion, most likely by removing excessive auxin to stabilize the hormone level in the feeding site. Our data also indicate a role of local auxin peaks in nematode attraction towards the root

Ectopic expression of Kip-related proteins restrains root-knot nematode-feeding site expansion

The development of nematode feeding sites induced by root-knot nematodes involves the synchronized activation of cell cycle processes such as acytokinetic mitoses and DNA amplification. A number of key cell cycle genes are reported to be critical for nematode feeding site development. However, it remains unknown whether plant cyclin-dependent kinase (CDK) inhibitors such as the Arabidopsis interactor/inhibitor of CDK (ICK)/Kip-related protein (KRP) family are involved in nematode feeding site development. This study demonstrates the involvement of Arabidopsis ICK2/KRP2 and ICK1/KRP1 in the control of mitosis to endoreduplication in galls induced by the root-knot nematode Meloidogyne incognita. ! Using ICK/KRP promoter-GUS fusions and mRNA in situ hybridizations, we showed that ICK2/KRP2, ICK3/KRP5 and ICK4/KRP6 are expressed in galls after nematode infection. Loss-of-function mutants have minor effects on gall development and nematode reproduction. Conversely, overexpression of both ICK1/KRP1 and ICK2/KRP2 impaired mitosis in giant cells and blocked neighboring cell proliferation, resulting in a drastic reduction of gall size. ! Studying the dynamics of protein expression demonstrated that protein levels of ICK2/ KRP2 are tightly regulated during giant cell development and reliant on the presence of the nematode. ! This work demonstrates that impeding cell cycle progression by means of ICK1/KRP1 and ICK2/KRP2 overexpression severely restricts gall development, leading to a marked limitation of root-knot nematode development and reduced numbers of offsprin

The plant WEE1 kinase is involved in checkpoint control activation in nematode-induced galls

Galls induced by plant‐parasitic nematodes involve a hyperactivation of the plant mitotic and endocycle machinery for their profit. Dedifferentiation of host root cells includes drastic cellular and molecular readjustments. In such background, potential DNA damage in the genome of gall cells is eminent. We questioned if DNA damage checkpoints activation followed by DNA repair occurred, or was eventually circumvented, in nematode‐induced galls. Galls display transcriptional activation of the DNA damage checkpoint kinase WEE1, correlated with its protein localization in the nuclei. The promoter of the stress marker gene SMR7 was evaluated under the WEE1‐knockout background. Drugs inducing DNA damage and a marker for DNA repair, PARP1 were used to understand mechanisms that might cope with DNA damage in galls. Our functional study revealed that gall cells lacking WEE1 conceivably entered mitosis prematurely disturbing the cell cycle despite the loss of genome integrity. The disrupted nuclei phenotype in giant cells hinted to the accumulation of mitotic defects. As well, WEE1‐knockout in Arabidopsis and downregulation in tomato repressed infection and reproduction of root‐knot nematodes. Together with data on DNA damaging drugs, we suggest a conserved function for WEE1 controlling a G1/S cell cycle arrest in response to replication defect in galls

Plant Cyclin-Dependent Kinase Inhibitors of the KRP Family: Potent Inhibitors of Root-Knot Nematode Feeding Sites in Plant Roots

Root-knot nematodes (RKN), Meloidogyne spp., are distributed worldwide and impose severe economic damage to many agronomically important crops. The plant cell cycle machinery is considered one of the pivotal components for the formation of nematode feeding sites (NFSs) or galls. These feeding sites contain five to nine hypertrophied giant cells (GC) resulting from developmental reprogramming of host root cells by this pathogen. GC undergo synchronous waves of mitotic activity uncoupled from cytokinesis giving rise to large multinucleate cells. As development of the NFS progresses, multiple rounds of DNA synthesis occur in the nuclei of GC, coupled with nuclear and cellular expansion. These cells are highly metabolically active and provide the nematode with nutrients necessary for its development and completion of its life cycle. In Arabidopsis seven cyclin dependent kinase inhibitors (CKIs) belonging to the interactors/inhibitors of the cyclin dependent kinases (ICK) family, also referred as Kip-Related Proteins (KRPs) have been identified. Interactions of KRPs with CDK/Cyclin complexes decrease CDK activity, affecting both cell cycle progression and DNA content in a concentration-dependent manner. We performed the functional analysis of all Arabidopsis KRP gene members during RKN interaction in Arabidopsis to obtain more insight into their role during gall development. We demonstrated that three members of this family (KRP2, KRP5, and KRP6) were highly expressed in galls and were important for cell cycle regulation during NFS development as shown by their different modes of action. We also pointed out that cell cycle inhibition through overexpression of all members of the KRP family can affect NFS development and consequently compromise the nematode’s life cycle. In this review we summarized our recent understanding of the KRP family of genes, and their role in controlling cell cycle progression at the RKN feeding site

O Fortalecimento da Cidadania como Estratégia de Design para Prevenção de Crimes

In addition to police action, other factors contribute to crime prevention, including design. The Crime Prevention through Environmental Design (CPTED) approach uses design to modify the offender's perception of the opportunity and benefits of crime. The goal of the research was to identify in Parque do Confisco (Belo Horizonte, Minas Gerais, Brazil), based on CPTED, factors that would stimulate criminal practice, as well as to verify if it would be possible to eliminate these factors. To this end, bibliographic research and spatial observation in the field were carried out. The analysis was based on the three strategies of CPTED: natural access control, natural surveillance and territorial reinforcement, as well as the investigation considered some aspects of the 3D approach (Designation, Definition and Design), typical of CPTED. The results revealed that low visibility, depredation and poor maintenance are the main aspects that stimulate criminal practice in the area. It is possible to implement improvements, however, it is necessary to build integrated projects that consider both physical and behavioral dimensions.Além da ação policial, outros fatores contribuem para a prevenção do crime, dentre eles, o design. A abordagem Crime Prevention Through Environmental Design (CPTED) utiliza o design para modificar a percepção do transgressor quanto à oportunidade e o benefício do crime. O objetivo da pesquisa consistiu em identificar no Parque do Confisco (município de Belo Horizonte, estado de Minas Gerais, Brasil), sob a ótica do CPTED, fatores que favoreceriam a prática criminosa, bem como verificar a possibilidade de supressão desses fatores. Para tanto, elaborou-se pesquisa bibliográfica e observação espacial em campo. A análise dos dados teve como base as três estratégias do CPTED: controle natural de acesso, vigilância natural e reforço territorial, bem como considerou alguns aspectos da abordagem 3D (Designação, Definição e Design), típica do CPTED. Os resultados revelaram que a baixa visibilidade, a depredação e a manutenção precária são os principais aspectos que favorecem a prática criminosa no local. É possível implantar melhorias, porém, faz-se necessária a construção de projetos integrados que considerem tanto as dimensões físicas quanto as comportamentais

The plant apoplasm is an important recipient compartment for nematode secreted proteins

Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode

The F-actin cytoskeleton in syncytia from non-clonal progenitor cells

The actin cytoskeleton of plant syncytia (a multinucleate cell arising through fusion) is poorly known: to date, there have only been reports about F-actin organization in plant syncytia induced by parasitic nematodes. To broaden knowledge regarding this issue, we analyzed F-actin organization in special heterokaryotic Utricularia syncytia, which arise from maternal sporophytic tissues and endosperm haustoria. In contrast to plant syncytia induced by parasitic nematodes, the syncytia of Utricularia have an extensive F-actin network. Abundant F-actin cytoskeleton occurs both in the region where cell walls are digested and the protoplast of nutritive tissue cells fuse with the syncytium and also near a giant amoeboid in the shape nuclei in the central part of the syncytium. An explanation for the presence of an extensive F-actin network and especially F-actin bundles in the syncytia is probably that it is involved in the movement of nuclei and other organelles and also the transport of nutrients in these physiological activity organs which are necessary for the development of embryos in these unique carnivorous plants. We observed that in Utricularia nutritive tissue cells, actin forms a randomly arranged network of F-actin, and later in syncytium, two patterns of F-actin were observed, one characteristic for nutritive cells and second—actin bundles—characteristic for haustoria and suspensors, thus syncytia inherit their F-actin patterns from their progenitors

Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction.

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Nematode effector proteins: an emerging paradigm of parasitism

Phytonematodes use a stylet and secreted effectors to modify host cells and ingest nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode effectors, with a particular focus on proteinaceous stylet-secreted effectors of sedentary endoparasitic phytonematodes, for which a wealth of information has surfaced in the past 10 yr. We provide an update on the effector repertoires of several of the most economically important genera of phytonematodes and discuss current approaches to dissecting their function. Lastly, we highlight the latest breakthroughs in effector discovery that promise to shed new light on effector diversity and function across the phylum Nematoda

On the use of dexamethasone-loaded liposomes to induce the osteogenic differentiation of human mesenchymal stem cells

Article first published online: 7 Oct. 2013Stem cells have received considerable attention by the scientific community because of their potential for tissue engineering and regenerative medicine. The most frequently used method to promote their differentiation is supplementation of the in vitro culture medium with growth/differentiation factors (GDFs). The limitations of that strategy caused by the short half-life of GDFs limit its efficacy in vivo and consequently its clinical use. Thus, the development of new concepts that enable the bioactivity and bioavailability of GDFs to be protected, both in vitro and in vivo, is very relevant. Nanoparticle-based drug delivery systems can be injected, protect the GDFs and enable spatiotemporal release kinetics to be controlled. Liposomes are well-established nanodelivery devices presenting significant advantages, viz. a high load-carrying capacity, relative safety and easy production, and a versatile nature in terms of possible formulations and surface functionalization. The main objective of the present study was to optimize the formulation of liposomes to encapsulate dexamethasone (Dex). Our results showed that the optimized Dex-loaded liposomes do not have any cytotoxic effect on human bone marrow-derived mesenchymal stem cells (hBMSCs). More importantly, they were able to promote an earlier induction of differentiation of hBMSCs into the osteogenic lineage, as demonstrated by the expression of osteoblastic markers, both phenotypically and genotypically. We concluded that Dex-loaded liposomes represent a viable nanoparticle strategy with enhanced safety and efficacy for tissue engineering and regenerative medicine.The authors thank the Portuguese Foundation for Science and Technology for a PhD grant (No. SFRH/BD/62465/2009, to N. S. Monteiro). This work was partly supported by the FIND and BIND Project (No. NMP4-SL-2009-229292) and the OsteoGraphy Project (No. PTDC/EME-MFE/2008)