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 polymerase chain reaction (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 kb. 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.

Assembly of the algal CO2-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif.

Approximately one-third of the Earth's photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid and how the organelle's subcompartments-membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath-are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we found that pyrenoid proteins share a sequence motif. We show that the motif is necessary and sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix-starch sheath interface suggests that the motif holds the pyrenoid's three subcompartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multilayered phase-separated organelle.

Thylakoid localized bestrophin-like proteins are essential for the CO2 concentrating mechanism of Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii possesses a CO2 concentrating mechanism (CCM) that helps in successful acclimation to low CO2 conditions. Current models of the CCM postulate that a series of ion transporters bring HCO3- from outside the cell to the thylakoid lumen, where the carbonic anhydrase 3 (CAH3) dehydrates accumulated HCO3- to CO2, raising the CO2 concentration for Ribulose bisphosphate carboxylase/oxygenase (Rubisco). Previously, HCO3- transporters have been identified at both the plasma membrane and the chloroplast envelope, but the transporter thought to be on the thylakoid membrane has not been identified. Three paralogous genes (BST1, BST2, and BST3) belonging to the bestrophin family have been found to be up-regulated in low CO2 conditions, and their expression is controlled by CIA5, a transcription factor that controls many CCM genes. YFP fusions demonstrate that all 3 proteins are located on the thylakoid membrane, and interactome studies indicate that they might associate with chloroplast CCM components. A single mutant defective in BST3 has near-normal growth on low CO2, indicating that the 3 bestrophin-like proteins may have redundant functions. Therefore, an RNA interference (RNAi) approach was adopted to reduce the expression of all 3 genes at once. RNAi mutants with reduced expression of BST1-3 were unable to grow at low CO2 concentrations, exhibited a reduced affinity to inorganic carbon (Ci) compared with the wild-type cells, and showed reduced Ci uptake. We propose that these bestrophin-like proteins are essential components of the CCM that deliver HCO3- accumulated in the chloroplast stroma to CAH3 inside the thylakoid lumen.

SUMO conjugation to the pattern recognition receptor FLS2 triggers intracellular signalling in plant innate immunity.

Detection of conserved microbial patterns by host cell surface pattern recognition receptors (PRRs) activates innate immunity. The FLAGELLIN-SENSITIVE 2 (FLS2) receptor perceives bacterial flagellin and recruits another PRR, BAK1 and the cytoplasmic-kinase BIK1 to form an active co-receptor complex that initiates antibacterial immunity in Arabidopsis. Molecular mechanisms that transmit flagellin perception from the plasma-membrane FLS2-associated receptor complex to intracellular events are less well understood. Here, we show that flagellin induces the conjugation of the SMALL UBIQUITIN-LIKE MODIFIER (SUMO) protein to FLS2 to trigger release of BIK1. Disruption of FLS2 SUMOylation can abolish immune responses, resulting in susceptibility to bacterial pathogens in Arabidopsis. We also identify the molecular machinery that regulates FLS2 SUMOylation and demonstrate a role for the deSUMOylating enzyme, Desi3a in innate immunity. Flagellin induces the degradation of Desi3a and enhances FLS2 SUMOylation to promote BIK1 dissociation and trigger intracellular immune signalling.

Comparison of the BBL mycobacteria growth indicator tube, the BACTEC 12B, and solid media for the isolation of Mycobacterium bovis.

We compared different methods for their ability to isolate Mycobacterium bovis from tissue samples from animals with lesions resembling bovine tuberculosis. In the first trial, M. bovis was isolated from 86 of 200 tissue samples that were cultured using 2 liquid media, BACTEC 12B and BBL mycobacteria growth indicator tube (MGIT), and a solid medium, Middlebrook 7H11 supplemented with pyruvate (7H11P). M. bovis was isolated from 2 samples with MGIT but not BACTEC 12B. M. bovis was isolated from 9 samples with BACTEC but not MGIT; these 9 samples came from the North Canterbury/Marlborough region of New Zealand. The proportion of tissues from which M. bovis was isolated with BACTEC 12B or MGIT and the mean time for isolation was different for samples from the North Canterbury/Marlborough region but not the rest of New Zealand. In the second trial, M. bovis was isolated from 401 of 1,033 tissues that were cultured using MGIT, Middlebrook 7H9 broth, or solid 7H11P. The proportion of isolates of M. bovis and the mean time for their isolation with MGIT was different for the North Canterbury/Marlborough and the rest of New Zealand. The reason for this difference was not determined but may be related to the genotypes present in this region. Genotyping using variable number tandem repeats (VNTRs) of 197 isolates of M. bovis revealed that the 44 isolates from North Canterbury/Marlborough were represented by 2 closely related VNTR types that were not found in 153 isolates from the remainder of New Zealand.

The ubiquitin conjugating enzyme, TaU4 regulates wheat defence against the phytopathogen Zymoseptoria tritici.

Mycosphaerella graminicola (Zymoseptoria tritici commonly known as Septoria), the causal agent of Septoria Leaf Blotch (STB), is considered one of the major threats to European wheat production. Previous studies have shown the importance of ubiquitination in plant defence against a multitude of pathogens. However the ubiquitination machinery in wheat is under studied, particularly E2 enzymes that have the ability to control the ubiquitination and thereby the fate of many different target proteins. In this study we identify an E2 enzyme, Triticum aestivum Ubiquitin conjugating enzyme 4 (TaU4) that functions in wheat defence against Septoria. We demonstrate TaU4 to be a bona fide E2 enzyme through an E2 charging assay. TaU4 localises in both the cytoplasm and nucleus, therefore potentially interacting with E3 ligases and substrate proteins in multiple compartments. Virus Induced Gene Silencing of TaU4 in wheat leaves resulted in delayed development of disease symptoms, reduced Septoria growth and reproduction. We conclude that TaU4 is a novel negative regulator of defence against Septoria.

Expression, Purification, and Enzymatic Analysis of Plant SUMO Proteases.

The conjugation of SUMO can profoundly change the behavior of substrate proteins, impacting a wide variety of cellular responses. SUMO proteases are emerging as key regulators of plant adaptation to its environment because of their instrumental role in the SUMO deconjugation process. Here, we describe how to express, purify, and determine SUMO deconjugation activity of a plant SUMO protease.

SUMO proteases: uncovering the roles of deSUMOylation in plants.

Plants have evolved to cope with changing environmental conditions. One way plants achieve this is through post-translational modification of target proteins by ubiquitination and SUMOylation. These post-translational modifiers (PMs) can alter stability, protein-protein interactions, and the overall fate of the protein. Both of these systems have remarkable similarities in terms of the process leading to attachment of the PM to its substrate : having to undertake activation, conjugation, and finally ligation to the target. In the ubiquitin system, there are a vast number of ubiquitin ligase enzymes (E3s) that provide specificity for the attachment of ubiquitin. With the SUMO system, only a small number of SUMO E3 ligases have so far been identified in the fully sequenced plant genomes. In Arabidopsis thaliana, there are only two SUMO E3s, compared to over 1400 ubiquitin E3s, a trend also observed in crop species such as Oryza sativa and Zea mays Recent research indicates that removing SUMO from its substrate by the enzymatically active SUMO proteases is a vital part of this system. A class of SUMO proteases called ubiquitin-like proteases (ULPs) are widespread in all eukaryotes; within plants, both monocot and dicot kingdoms have conserved and divergent ULPs and ULP-like proteases. This paper examines the roles ULPs have in stress responses and highlights the 'fine-tuning' of SUMO attachment/removal in balancing growth versus stress.

SUMO Is a Critical Regulator of Salt Stress Responses in Rice.

SUMO (Small Ubiquitin-like Modifier) conjugation onto target proteins has emerged as a very influential class of protein modification systems. SUMO1/2 double mutant plants are nonviable, underlining the importance of SUMO conjugation to plant survival. Once covalently bound, SUMO can alter a conjugated protein's stability and/or function. SUMO conjugation is a highly dynamic process that can be rapidly reversed by the action of SUMO proteases. The balance between the conjugated/deconjugated forms is a major determinant in the modulation of SUMO-target function. Despite the important mechanistic role of SUMO proteases in model plants, until now the identity or the function of these regulatory enzymes has not been defined in any crop plant. In this report, we reveal the ubiquitin-like protease class of SUMO protease gene family in rice (Oryza sativa) and demonstrate a critical role for OsOTS1 SUMO protease in salt stress. OsOTS-RNAi rice plants accumulate high levels of SUMO-conjugated proteins during salt stress and are highly salt sensitive; however, in non-salt conditions, they are developmentally indistinguishable from wild-type plants. Transgenic rice plants overexpressing OsOTS1 have increased salt tolerance and a concomitant reduction in the levels of SUMOylated proteins. We demonstrate that OsOTS1 confers salt tolerance in rice by increasing root biomass. High salinity triggers OsOTS1 degradation, indicating that increased SUMO conjugation in rice plants during salt stress is in part achieved by down-regulation of OTS1/2 activity. OsOTS1 is nuclear localized indicating a direct requirement of OsOTS1-dependent deSUMOylation activity in rice nuclei for salt tolerance.

The Power of Humility.

Consistently high-performing health systems and hospitals have one key trait in common: humility.

Ubiquitination in plant nutrient utilization.

Ubiquitin (Ub) is well-established as a major modifier of signaling in eukaryotes. However, the extent to which plants rely on Ub for regulating nutrient uptake is still in its infancy. The main characteristic of ubiquitination is the conjugation of Ub onto lysine residues of acceptor proteins. In most cases the targeted protein is rapidly degraded by the 26S proteasome, the major proteolysis machinery in eukaryotic cells. The Ub-proteasome system is responsible for removing most abnormal peptides and short-lived cellular regulators, which, in turn, control many processes. This allows cells to respond rapidly to intracellular signals and changing environmental conditions. This perspective will discuss how plants utilize Ub conjugation for sensing environmental nutrient levels. We will highlight recent advances in understanding how Ub aids nutrient homeostasis by affecting the trafficking of membrane bound transporters. Given the overrepresentation of genes encoding Ub-metabolizing enzymes in plants, intracellular signaling events regulated by Ub that lead to transcriptional responses due to nutrient starvation is an under explored area ripe for new discoveries. We provide new insight into how Ub based biochemical tools can be exploited to reveal new molecular components that affect nutrient signaling. The mechanistic nature of Ub signaling indicates that dominant form of any new molecular components can be readily generated and are likely shed new light on how plants cope with nutrient limiting conditions. Finally as part of future challenges in this research area we introduce the newly discovered roles of Ub-like proteins in nutrient homeostasis.

Identification of the domains of cauliflower mosaic virus protein P6 responsible for suppression of RNA silencing and salicylic acid signalling.

Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.

Sustained protection against tuberculosis conferred to a wildlife host by single dose oral vaccination.

BACKGROUND: Vaccination of wildlife against bovine tuberculosis (TB) is being considered by several countries to reduce the transmission of Mycobacterium bovis infection to livestock. In New Zealand, where introduced brushtail possums (Trichosurus vulpecula) are the major wildlife hosts, we have previously shown that repeat applications of a lipid-encapsulated oral bacille Calmette-Guerin (BCG) vaccine reduce the incidence of naturally acquired TB in wild possums. Here we extend this conceptual demonstration to an operational level, assessing long-term protection against TB conferred to free-living possums by a single oral immunisation. METHODS: Possums in a non-TB area were randomly allocated to receive lipid-formulated BCG vaccine or remained unvaccinated. After initial trials to assess vaccine immunogenicity and establishment of protection within the first year post-vaccination, 13 individuals of each treatment group were relocated to a biosecurity facility and challenged (at 28 months post-vaccination) by subcutaneous injection of virulent M. bovis. RESULTS: Vaccine immunogenicity and short-term protection were confirmed at 2 months and 12 months post-vaccination, respectively. In the long-term assessment, vaccinated possums had significantly reduced bacterial counts in peripheral lymph nodes compared to controls, with 0.6-2.3 log(10)-fold reductions in M. bovis burdens. DISCUSSION: The magnitude of protective response by possums to experimental challenge at 28 months post-vaccination is known to equate to a high degree of protection against natural infection in this species. With techniques for oral bait delivery well advanced, the longevity of protection demonstrated here shows that an operable wildlife vaccine against TB is feasible.

More quality measures versus measuring what matters: a call for balance and parsimony.

External groups requiring measures now include public and private payers, regulators, accreditors and others that certify performance levels for consumers, patients and payers. Although benefits have accrued from the growth in quality measurement, the recent explosion in the number of measures threatens to shift resources from improving quality to cover a plethora of quality-performance metrics that may have a limited impact on the things that patients and payers want and need (ie, better outcomes, better care, and lower per capita costs). Here we propose a policy that quality measurement should be: balanced to meet the need of end users to judge quality and cost performance and the need of providers to continuously improve the quality, outcomes and costs of their services; and parsimonious to measure quality, outcomes and costs with appropriate metrics that are selected based on end-user needs.

Transmission of Mycobacterium orygis (M. tuberculosis complex species) from a tuberculosis patient to a dairy cow in New Zealand.

Mycobacterium orygis, previously called the oryx bacillus, is a member of the Mycobacterium tuberculosis complex and has been reported only recently as a cause of human tuberculosis in patients of South Asian origin. We present the first case documenting the transmission of this organism from a human to a cow.

Controlling healthcare costs by removing waste: what American doctors can do now.

Healthcare costs are unsustainable. The authors propose a solution to control costs without rationing (deliberate withholding of effective care) or payment reductions to doctors and hospitals. Three physician-led strategies comprise this solution: reduce (1) overuse of health services, (2) preventable complications and (3) waste within healthcare processes. These challenges know no borders.

Isolation of Mycobacterium bovis and other mycobacterial species from ferrets and stoats.

As part of wildlife surveillance for bovine tuberculosis, pooled lymph nodes from 21,481 ferrets, 1056 stoats and 83 weasels were cultured for mycobacteria. A total of 268 isolates of Mycobacterium bovis were obtained from ferrets, 2 from stoats and none from weasels, demonstrating the presence of a wildlife reservoir of infection in ferrets. DNA typing by restriction endonuclease analysis (REA) of 48 selected isolates of M. bovis revealed 23 REA types. Twenty-one of these types had previously been isolated from cattle and farmed deer, demonstrating a complex cycle of infection involving wildlife and domestic animals. Apart from M. bovis, a further 208 mycobacterial isolates were obtained, the majority of which (178) were members of the M. avium complex. Speciation of the remaining 30 mycobacterial isolates by DNA sequencing of the 16s rRNA gene, identified half the isolates as M. triplex. Other species identified included M. fortuitum, M. florentinum, M. interjectum, M. intracellulare, M. holsaticum, and M. septicum/M. peregrinum.