Host Plant Cells Research Articles

<I>Cymadothea trifolii</I>, an obligate biotrophic leaf parasite of <I>Trifolium</I>, belongs to <I>Mycosphaerellaceae</I> as shown by nuclear ribosomal DNA analyses

The ascomycete Cymadothea trifolii, a member of the Dothideomycetes, is unique among obligate biotrophic fungi in its capability to only partially degrade the host cell wall and in forming an astonishingly intricate interaction apparatus (IA) in its own hyphae, while the attacked host plant cell is triggered to produce a membranous bubble opposite the IA. However, no sequence data are currently available for this species. Based on molecular phylogenetic results obtained from complete SSU and partial LSU data, we show that the genus Cymadothea belongs to the Mycosphaerellaceae (Capnodiales, Dothideomycetes). This is the first report of sequences obtained for an obligate biotrophic member of Mycosphaerellaceae.

VirE2: A Unique ssDNA-Compacting Molecular Machine

The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes.

Growth and Nitrogen Fixation of Different Medicago Sativa-Sinorhizobium Meliloti Associations Under Conditions of Mineral Elements Shortage

ABSTRACTSinorhizobium meliloti is an α-proteobacteria of the family Rhizobiaceae that alternates between a free-living phase in soil and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. The present study was designed to compare the difference in responses to nitrogen and carbon limitation in free living Sinorhizobium meliloti strains and their ability to form symbiotic association with alfalfa (Medicago sativa) plants. The effectiveness of observed symbiotic associations was evaluated by number of formed nodules, nitrogen fixing activity, and plant biomass production in control environmental conditions of a growth chamber. A wild type strain Sinorhizobium meliloti 1021 and two mutants—Sinorhizobium meliloti NitR and Sinorhizobium meliloti TspO were used in this research. Both mutant strains were previously created to study the general stress response in Sinorhizobium meliloti and its regulation mechanisms. The products of the genes named tspO and nitR, act either as direct or indirect regulators of gene expression in response to various stresses, including starvation. The up- and down regulated genes under conditions of nitrogen and carbon limitation were identified in free living forms of the three strains. Comparison of the genes differentially expressed in the wild type strain to those found in the tspO and nitR mutants showed no significant difference. In both starvation conditions the most effective symbiotic system was established between alfalfa and Sinorhizobium meliloti TspO, concerning nitrogen fixing capacity and plant biomass production.

AY-WB Phytoplasma Secretes a Protein That Targets Plant Cell Nuclei

The fully sequenced genome of aster yellows phytoplasma strain witches' broom (AY-WB; Candidatus Phytoplasma asteris) was mined for the presence of genes encoding secreted proteins based on the presence of N-terminal signal peptides (SP). We identified 56 secreted AY-WB proteins (SAP). These SAP are candidate effector proteins potentially involved in interaction with plant and insect cell components. One of these SAP, SAP11, contains an N-terminal SP sequence and a eukaryotic bipartite nuclear localization signal (NLS). Transcripts for SAP11 were detected in AY-WB-infected plants. Yellow fluorescence protein (YFP)-tagged SAP11 accumulated in Nicotiana benthamiana cell nuclei, whereas the nuclear targeting of YFP-tagged SAP11 mutants with disrupted NLS was inhibited. The nuclear transport of YFP-SAP11 was also inhibited in N. benthamiana plants in which the expression of importin alpha was knocked down using virus-induced gene silencing (VIGS). Furthermore, SAP11 was detected by immunocytology in nuclei of young sink tissues of China aster plants infected with AY-WB. In summary, this work shows that AY-WB phytoplasma produces a protein that targets the nuclei of plant host cells; this protein is a potential phytoplasma effector that may alter plant cell physiology.

Characterization and expression analysis of a gene encoding a secreted lipase-like protein expressed in the salivary glands of the larval Hessian fly, Mayetiola destructor (Say)

In a salivary gland transcriptomics study we identified a cDNA with a full-length open reading frame for a gene ( MdesL1) encoding a lipase-like protein expressed in the salivary glands of the larval Hessian fly, Mayetiola destructor (Say). Fluorescent in situ hybridization on salivary polytenes positioned MdesL1 on the long arm of Autosome 1. BLASTp and conserved domain searches revealed the deduced amino acid sequence contained a lipase superfamily domain with similarity to lipases and phospholipases from other insects. A secretion signal peptide was identified at the amino terminus of the deduced amino acid sequence. Analysis of the transcript of MdesL1 in larval Hessian fly tissues by quantitative real-time PCR (qPCR) revealed the greatest abundance was in salivary glands. Analysis of transcript levels during development showed the greatest level was detected in feeding 1st-instar and early 2nd-instar larvae. Transcript levels increased dramatically over time in larvae feeding on susceptible wheat but were detected at low levels in larvae feeding on resistant wheat. These data suggest the protein encoded by MdesL1 is likely secreted into host-plant cells during larval feeding and could be involved in extra-oral digestion and changes in host-cell permeability or in generating a second messenger in a host-cell-signaling cascade.

Class III peroxidases in plant defence reactions

When plants are attacked by pathogens, they defend themselves with an arsenal of defence mechanisms, both passive and active. The active defence responses, which require de novo protein synthesis, are regulated through a complex and interconnected network of signalling pathways that mainly involve three molecules, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), and which results in the synthesis of pathogenesis-related (PR) proteins. Microbe or elicitor-induced signal transduction pathways lead to (i) the reinforcement of cell walls and lignification, (ii) the production of antimicrobial metabolites (phytoalexins), and (iii) the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Among the proteins induced during the host plant defence, class III plant peroxidases (EC 1.11.1.7; hydrogen donor: H(2)O(2) oxidoreductase, Prxs) are well known. They belong to a large multigene family, and participate in a broad range of physiological processes, such as lignin and suberin formation, cross-linking of cell wall components, and synthesis of phytoalexins, or participate in the metabolism of ROS and RNS, both switching on the hypersensitive response (HR), a form of programmed host cell death at the infection site associated with limited pathogen development. The present review focuses on these plant defence reactions in which Prxs are directly or indirectly involved, and ends with the signalling pathways, which regulate Prx gene expression during plant defence. How they are integrated within the complex network of defence responses of any host plant cell will be the cornerstone of future research.

Role of vitronectin-like protein in Agrobacterium attachment and transformation of Arabidopsis cells

The role of plant vitronectin-like protein (Vn) in Agrobacterium-host plant interactions and receptor-specific bacterial attachment is unclear and still open to debate. Using a well-established Agrobacterium-mediated Arabidopsis transformation system, the marker gene beta-glucuronidase (GUS) of Escherichia coli, and biochemical and cytological methods, such as ELISA tests, immunoblots, immunolocalization, and functional in vitro binding assays, we have reassessed the role of Vn in receptor-specific bacterial attachment and transformation. We provide evidence that Vn is present in the host plant cells and anti-human vitronectin antibody cross-reacts with a 65-kDa protein from Arabidopsis cells. The specificity of the immunological cross-reactivity of anti-vitronectin antibodies was further demonstrated by ELISA competition experiments. Immunogold labeling showed that Vn is localized in the plant cell wall, and its level increased considerably after phytohormone treatment of the petiole explants. However, Agrobacterium attachment was unaffected, and no inhibition of petiole cell transformation was detected in the presence of human vitronectin and anti-vitronectin antibodies in the media. Additionally, no correlation between the occurrence of Vn, attachment of bacteria to the cells, and susceptibility to Agrobacterium-mediated transformation was observed. Taken together, our data do not support a functional role of plant Vn as the receptor for site-specific Agrobacterium attachment leading to the transformation of Arabidopsis cells.

Moving targets: rapid evolution of oomycete effectors

Plant pathogenic microbes secrete proteins known as effectors, which enter the cytoplasm of plant cells and suppress host defences. Known effectors in oomycete pathogens possess an RXLR-EER motif in their amino acid sequence that is necessary for transport of the effector into a host plant cell. A large number of putative effectors have now been identified in oomycete genomes, the sequences of which show evidence of diversifying selection at their C terminus. Here, we describe recent progress in characterizing RXLR-EER effectors and discuss why so many of these rapidly evolving proteins are encoded by the genomes of plant pathogenic oomycetes.

Expression of prune dwarf Ilarvirus coat protein sequences in Nicotiana benthamiana plants interferes with PDV systemic proliferation

Prune dwarf virus (PDV) is an Ilarvirus systemically infecting almond trees and other Prunus species and spreading through pollen, among other means. We have studied strategies based on coat protein (cp) gene to block PDV replication in host plant cells. A Portuguese isolate of PDV was obtained from infected almond leaves and used to produce the cDNA of the cp gene. Various constructs were prepared based on this sequence, aiming for the transgenic expression of the original or modified PDV coat protein (cpPDVSense and cpPDVMutated) or for the expression of cpPDV RNA (cpPDVAntisense and cpPDVwithout start codon). All constructs were tested in a PDV host model, Nicotiana benthamiana, and extensive molecular characterization and controlled infections were performed on transformants and their progenies. Transgenic plants expressing the coat protein RNA were able to block the proliferation of a PDV isolate sharing only 91% homology with the isolate used for cpPDV cloning, as evaluated by DAS-ELISA on newly developed leaves. With cp expression, the blockage of PDV proliferation in newly developed leaves was only achieved with the construct cpPDV Mutated, where the coat protein has a substitution in the 14th aa residue, with arginine replaced by alanine. This result points to a possible role of the mutated amino acid in the virus ability to replicate and proliferate. This work reveals the possibility of achieving protection against PDV through either coat protein RNA or mutated cp sequence.

VirE2: A Unique ssDNA-Compacting Molecular Machine

The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.

Geminivirus resistance breeding in common bean.

Abstract Common bean ( Phaseolus vulgaris L.) is among the most important food staples worldwide, mainly because of its relatively high protein content and other valuable nutritional qualities. Unfortunately, common beans are highly susceptible to plant viruses in general, including one of the largest families of plant viruses known as the Geminiviridae . These viruses, unlike most RNA plant viruses, possess a DNA genome that allows these pathogens to replicate in the nucleus of their plant host cells, thus, causing major damage to susceptible legume crops. Although some geminiviruses, belonging to two ( Mastrevirus and Curtovirus ) of the four genera that compose the Geminiviridae , can be transmitted by leafhoppers (Cicadellidae) to common bean, most geminiviruses that attack legumes belong to the genus Begomovirus , and are transmitted by the whitefly Bemisia tabaci (Genn.). In fact, the type species of this genus is bean golden yellow mosaic virus (BGYMV), which attacks over a million hectares of common bean grown in the lowlands and mid-altitude valleys of Central America, southern Mexico, the Caribbean region and northern South America. Another begomovirus, bean golden mosaic virus (BGMV), affects over two million hectares of common bean in Brazil, northern Argentina and the lowlands of Bolivia, in South America. In this region, bean dwarf mosaic virus (BDMV) has also caused the loss of thousands of hectares of highly valuable common bean genotypes in northwestern Argentina. In northwestern Mexico, thousands of hectares of common bean have been under the constant attack of a begomovirus related to a virus originally described in cucurbits, squash leaf curl virus (SLCV), in southwestern USA, currently known in Mexico as bean calico mosaic virus (BCaMV). Fortunately, none of these viruses is present in Eastern Africa, where the common bean is also a major food staple. Another Old World begomovirus reported to attack common bean both in the Old and New World, is tomato yellow leaf curl virus (TYLCV). This review describes the different breeding approaches followed since the 1970s to introduce resistance to begomoviruses infecting important legume crops, particularly common bean. This has been a challenging process, considering that no immune genotypes have been identified in more than 30000 common bean genotypes exposed to begomoviruses, such as BGMV or BGYMV.

Involvement of polyamines in the root nodule regulation of soybeans (Glycine max)

In leguminous plants, infection with rhizobia leads to the formation of root nodules that fix atmospheric nitrogen and supply it as ammonium to the host plant cells. The formation of nitrogen-fixing nodules is nutritionally benefi- cial, but excessive nodule production appears to be detrimental to the host legumes because of the resultant over-consumption of photosynthetic products. Therefore, the number of root nodules in leguminous plants is tightly regulated by shoot-root signaling known as a feedback or autoregulation of nodulation. Recently, we found that foliar application of polyamines and an effective inhibitor of polyamine biosynthesis regulates the number of nodules in soybean plants. In this review, we discuss the possible function of polyamines as a systemic regulator of nodule formation.

Differential Effects of Phenylalanine Ammonia Lyase, Cinnamyl Alcohol Dehydrogenase, and Energetic Metabolism Inhibition on Resistance of Appropriate Host and Nonhost Cereal–Rust Interactions

ABSTRACT Effects of phenylpropanoid and energetic metabolism inhibition on resistance were studied during appropriate host and nonhost cereal-rust interactions. In the appropriate barley-Puccinia hordei interaction, phenylalanine ammonia lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) inhibition reduced penetration resistance in two genotypes, suggesting a role for phenolics and lignins in resistance. Interestingly, penetration resistance of the barley genotype 17.5.16 was not affected by phenylpropanoid biosynthesis but penetration resistance was almost completely inhibited by D-mannose, which reduces the energy available in plant host cells. This suggests a parallel in the cellular basis of penetration resistance between 17.5.16 rust and mlo barleys powdery mildew interaction. Results revealed differing patterns of programmed cell death (PCD) in appropriate versus nonhost rust interactions. PAL and CAD inhibitors reduced PCD (hypersensitivity) in appropriate interactions. Conversely, they had no effect in PCD of wheat to P. hordei; whereas D-mannose dramatically reduced nonhost resistance and allowed colony establishment. The differential effects of inhibitors in the expression of the different resistances and the commonalities with the cereal-powdery mildew interaction is analyzed and discussed.

The rules of engagement

The rules of engagement

The Best Offense Is Someone Else’s Defense

Part of the strategy that Agrobacterium uses to attack its host plant cells includes transferring part of its genome into the plant cell’s genome. This DNA transfer event has been effectively used as a tool for plant genetic transformation in the lab. In the natural interaction, Agrobacterium uses a complex of proteins, including both its own and the host’s, to effect the transfer of its DNA into the plant cell nucleus. One of the plant’s proteins, VIP1, was known to function for Agrobacterium DNA transfer, but its use to the host plant was unknown. Djamei et al. have now discovered that the plant’s VIP1 is supposed to function in a pathogen defense cascade. Thus, Agrobacterium derails the plant’s defense response by using one of the plant’s own effectors to carry the bacterial DNA into the plant nucleus. A. Djamei, A. Pitzschke, H. Nakagami, I. Rajh, H. Hirt, Trojan horse strategy in Agrobacterium transformation: Abusing MAPK defense signaling. Science 318 , 453-456 (2007). [Abstract] [Full Text]

The role of primary germ tubes in the life cycle of Blumeria graminis: The primary germ tube is responsible for the suppression of resistance induction of a host plant cell

When the conidia of Blumeria graminis f. sp. hordei (Bgh) are inoculated on barley coleoptile cells they produce short germ tubes called primary germ tubes (PGTs) about 2 h after inoculation. We evaluated the positive role of a PGT in inducing accessibility of the host cell under the germ tube. When an appressorium (APP) penetrated the same cell on which a PGT was present, the ratio of haustorium formation (penetration efficiency) was significantly higher than when an APP penetrated the cell adjacent to the one on which a PGT was present. When an APP penetrated the cell laterally adjacent to the one on which a PGT was present we killed the cell under the PGT by puncturing it with a microneedle and then investigated the penetration efficiency of the cell adjacent to the dead cell. As a control we killed the cell longitudinally adjacent to the one on which a PGT was present and investigated the penetration efficiency of the laterally adjacent cell. The results showed that the penetration efficiency of the former was significantly lower than that of the latter. This suggests that some accessibility factor might transfer from a cell on which a PGT is present to a laterally adjacent cell. The existence of a conidium body but not a PGT was not effective for induced accessibility of the host cell. Moreover, when a Bgh germling was removed 6 h after inoculation and another germling was transferred to the same cell, the penetration efficiency was significantly higher than that of control. As a control, a Bgh germling was transferred to a cell on which no germling was present. These results suggest that the existence of PGT is effective for induced accessibility of a host cell when penetrated by Bgh. However, it is unclear whether or not a PGT secretes some substance(s) which suppresses the resistance induction of a host cell.

A translocation signal for delivery of oomycete effector proteins into host plant cells

Bacterial, oomycete and fungal plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK--representing a change that conserves physicochemical properties of the protein--P. infestans fails to deliver Avr3a or an Avr3a-GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.

Combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala(40)-Val(59)-Phe(75)-Ser(130)-Met(184) or Ser(40)-Leu(59)-Tyr(75)-Thr(130)-Leu(184)) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala(40) and Phe(75) or Ser(40) and Tyr(75)) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala(40) to Ser(40) or Phe(75) to Tyr(75)) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

Characterization of the N-terminal segment used by the barley yellow dwarf virus movement protein to promote interaction with the nuclear membrane of host plant cells

The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic α-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be α-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5–1.0 cm −1) and induced surface pressure changes of 2 mN m −1 in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic α-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.

The AGC Kinase MtIRE

The development of nitrogen fixing root nodules is complex and involves an interplay of signaling processes. During maturation of plant host cells and their endocytosed rhizobia in symbiosomes, host cells and symbiosomes expand. This expansion is accompanied by a large quantity of membrane biogenesis. We recently characterized an AGC kinase gene, MtIRE, that could play a role in this expansion. MtIRE’s expression coincides with host cell and symbiosome expansion in the proximal side of the invasion zone in developing Medicago truncatula nodules. MtIRE’s closest homolog is the Arabidopsis AGC kinase family IRE gene, which regulates root hair elongation. AGC kinases are regulated by phospholipid signaling in animals and fungi as well as in the several instances where they have been studied in plants. Here we suggest that a phospholipid signaling pathway may also activate MtIRE activity and propose possible upstream activators of MtIRE protein’s presumed AGC kinase activity.