Cutaneous 5T cell lymphoma (CTCL), characterized by malignant T cells infiltrating the skin with potential for dissemination, remains a challenging disease to diagnose and treat due to disease heterogeneity, treatment resistance, and lack of effective and standardized diagnostic and prognostic clinical tools. Currently, diagnosis of CTCL practically relies on clinical presentation, histopathology, and immunohistochemistry. These methods are collectively fraught with limitations in sensitivity and specificity. Fortunately, recent advances in flow cytometry, polymerase chain reaction, high throughput sequencing, and other molecular techniques have shown promise in improving diagnosis and treatment of CTCL. Examples of these advances include T cell receptor clonotyping via sequencing to detect CTCL earlier in the disease course and single-cell RNA sequencing to identify gene expression patterns that commonly drive CTCL pathogenesis. Experience with these techniques has afforded novel insights which may translate into enhanced diagnostic and therapeutic approaches for CTCL.

Circulating memory CD8 T cell trafficking and protective capacity during liver-stage malaria infection remains undefined. We find that effector memory CD8 T cells (Tem) infiltrate the liver within 6 hours after malarial or bacterial infections and mediate pathogen clearance. Tem recruitment coincides with rapid transcriptional upregulation of inflammatory genes in Plasmodium-infected livers. Recruitment requires CD8 T cell-intrinsic LFA-1 expression and the presence of liver phagocytes. Rapid Tem liver infiltration is distinct from recruitment to other non-lymphoid tissues in that it occurs both in the absence of liver tissue resident memory "sensing-and-alarm"function and -42 hours earlier than in lung infection by influenza virus. These data demonstrate relevance for Tem in protection against malaria and provide generalizable mechanistic insights

Radiation-attenuated sporozoite (RAS) vaccination offers hope for global malaria control through induction of protective liver-stage-specific memory CD8 T cells. Effective RAS vaccination regimens exist; however, widespread implementation remains unfeasible. A key difficulty resides in the need to administer three or more doses i.v. to achieve sufficient immunity. Strategies to reduce the number of RAS doses are therefore desirable. Here we used mice to model human immune responses to a single, suboptimal weight-normalized RAS dose administered i.v. followed by subunit vaccination to amplify liver-stage-specific memory CD8 T cells. RAS+subunit prime-boost regimens increased the numbers of liver-stage-specific memory CD8 T cells to a level greater than is present after one RAS vaccination. Both i.v. and i.m. subunit vaccine delivery induced immunity in mice, and many vaccinated mice completely cleared liver infection. These findings are particularly relevant to human vaccine development because RAS+subunit prime-boost vaccination would reduce the logistical challenges of multiple RAS-only immunizations.

New research demonstrates that γδ T cells recognize Plasmodium -infected erythrocytes via interaction of the T cell antigen receptor with the phosphoantigen sensor BTN3A1 and subsequently destroy infected cells through either cytotoxic molecule secretion or antibody-dependent phagocytosis.

Each year over 200 million malaria infections occur, with over 400 000 associated deaths. Vaccines formed with attenuated whole parasites can induce protective memory CD8 T cell responses against liver-stage malaria; however, widespread administration of such vaccines is logistically chap lenging. Recent scientific findings are delineating how protective memory CD8 T cell populations are primed and maintained and how such cells mediate immunity to liver-stage malaria. Memory CD8 T cell anatomic localization and expression of transcription factors, homing receptors, and signaling molecules appear to play integral roles in protective immunity to fiver-stage malaria Further investigation of how such factors contribute to optimal protective memory CD8 T cell generation and maintenance in humans will inform efforts for improved vaccines.

Each year over 200 million malaria infections occur, with over 400 000 associated deaths. Vaccines formed with attenuated whole parasites can induce protective memory CD8 T cell responses against liver-stage malaria; however, widespread administration of such vaccines is logistically challenging. Recent scientific findings are delineating how protective memory CD8 T cell populations are primed and maintained and how such cells mediate immunity to liver-stage malaria. Memory CD8 T cell anatomic localization and expression of transcription factors, homing receptors, and signaling molecules appear to play integral roles in protective immunity to liver-stage malaria. Further investigation of how such factors contribute to optimal protective memory CD8 T cell generation and maintenance in humans will inform efforts for improved vaccines.

It is hard to overstate the importance of flow cytometry for immunology research. As the field has advanced, the need for an array of fluorophores to meet different excitation and emission characteristics has increased. A predominant method of extending the range of available fluorophores is through addition of tandem conjugates to existing primary fluorophores (e.g., allophycocyanin [APC] fluorophore with a Cy7 conjugate yielding APC-Cy7) (Gerstner et al., 2002Gerstner A.O.H. Lenz D. Laffers W. Hoffman R.A. Steinbrecher M. Bootz F. Tárnok A. Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry.Cytometry. 2002; 48: 115-123Crossref PubMed Scopus (48) Google Scholar). This ever-broadening collection of tandem fluorophores has allowed further definition of numerous cell populations and are so often utilized that they are now integral to developing suitable flow cytometry antibody panels, particularly with the advent of modern flow spectral cytometers capable of detecting >30 distinct fluorophores (Cytek, 2020CytekCytek Aurora.https://cytekbio.com/pages/aurora#tab-dataDate: 2020Google Scholar). Tandem conjugates (e.g., Cy7) are activated through Förster resonance energy transfer (FRET) from its primary fluorophore and subsequently alter the emission spectra of the primary fluorophore (Gerstner et al., 2002Gerstner A.O.H. Lenz D. Laffers W. Hoffman R.A. Steinbrecher M. Bootz F. Tárnok A. Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry.Cytometry. 2002; 48: 115-123Crossref PubMed Scopus (48) Google Scholar). However, tandem dyes can degrade and/or lose their FRET capacity, leading to emission in the primary fluorophore's spectrum (Figure S1A; e.g., APC-Cy7 degrades into APC) (Maecker et al., 2004Maecker H.T. Frey T. Nomura L.E. Trotter J. Selecting fluorochrome conjugates for maximum sensitivity.Cytometry A. 2004; 62: 169-173Crossref PubMed Scopus (160) Google Scholar). Tandem degradation is a well-established phenomenon, with several studies exploring how different staining conditions (e.g., light, temperature, cell fixation, presence of serum in staining buffer) contribute to this degradation (Le Roy et al., 2009Le Roy C. Varin-Blank N. Ajchenbaum-Cymbalista F. Letestu R. Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.Cytometry A. 2009; 75: 882-890Crossref PubMed Scopus (30) Google Scholar, Morawski et al., 2019Morawski P.A. Motley S.J. Campbell D.J. Rapid Light-Dependent Degradation of Fluorescent Dyes in Formulated Serum-Free Media.Immunohorizons. 2019; 3: 585-592Crossref PubMed Scopus (4) Google Scholar). Establishing the level of degradation and fluorescence overlap is part of the reason instrument compensation prior to using tandem fluorophores is critical. Yet, compensation relies on the assumption of equivalent cell conditions between samples and experimental groups. We encountered a substantial problem with this assumption. Specifically, individual sample variability could influence the degree of tandem degradation, thereby variably impacting the signal of the primary fluorochrome on a given cell population. The extent of tandem dye degradation can substantially alter a researcher's interpretation of their data if this "phantom" signal, as a result of tandem degradation, is believed to represent a different protein's expression. When utilizing the cecal ligation and puncture (CLP) model of sepsis induction (Jensen et al., 2018Jensen I.J. Sjaastad F.V. Griffith T.S. Badovinac V.P. Sepsis-Induced T Cell Immunoparalysis: The Ins and Outs of Impaired T Cell Immunity.J. Immunol. 2018; 200: 1543-1553PubMed Google Scholar), we consistently observed aberrant results when multiple cell subsets were compared using tandem conjugates in CLP and sham (control) hosts. These oddities were determined to be due to degradation of an APC-efluor780 tandem to elicit a phantom APC signal detectable in the APC channel when no single APC-labeled antibodies are included (Figure S1B). Few APC+APC-Cy7− cells were observed (data not shown), suggesting that degradation of all tandems on a cell is rare, supporting our model (Figure S1A). If an APC-labeled antibody had been present, this would have impacted the interpretation of the APC antibody target's expression. Using cells only labeled by a tandem fluorophore, this phenomenon was observed across multiple cytometers (data not shown) and occurred regardless of tandem type or antibody target (Figure S1C). Given that the staining conditions for the two experimental groups were identical we reasoned that some biochemical feature(s) of CLP-induced sepsis must be affecting the cells to more potently degrade fluorophore tandems. Similar to Le Roy et al., we observed that oxidation, through addition of H2O2 to the staining buffer, promotes tandem degradation (Figure S1D) (Le Roy et al., 2009Le Roy C. Varin-Blank N. Ajchenbaum-Cymbalista F. Letestu R. Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.Cytometry A. 2009; 75: 882-890Crossref PubMed Scopus (30) Google Scholar). To address whether the sepsis-induced tandem degradation was due to oxidation, 2-mercaptoethanol (BME, a potent reducing agent) was added to the staining buffer (Figure S1E) and was sufficient to reduce tandem degradation, similar to the use vitamin C for the same objective (Le Roy et al., 2009Le Roy C. Varin-Blank N. Ajchenbaum-Cymbalista F. Letestu R. Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.Cytometry A. 2009; 75: 882-890Crossref PubMed Scopus (30) Google Scholar, Morawski et al., 2019Morawski P.A. Motley S.J. Campbell D.J. Rapid Light-Dependent Degradation of Fluorescent Dyes in Formulated Serum-Free Media.Immunohorizons. 2019; 3: 585-592Crossref PubMed Scopus (4) Google Scholar). Granulocytes have a greater degree of tandem degradation than lymphocytes (Le Roy et al., 2009Le Roy C. Varin-Blank N. Ajchenbaum-Cymbalista F. Letestu R. Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.Cytometry A. 2009; 75: 882-890Crossref PubMed Scopus (30) Google Scholar), and we observed a trending positive correlation between granulocyte frequency and degree of tandem degradation (data not shown). These observations, along with an increased proportion of granulocytes in CLP samples (data not shown), suggested that reactive oxygen species (ROS) may be the cause of the differential tandem degradation. Indeed, we observed elevated ROS production by cells from CLP samples relative to control (Sham) (Figures S1E and S1G). To determine whether this occurs in other highly inflammatory infections, mice were infected with non-lethal blood-stage Plasmodium yoelii 17XNL (PyXNL) (malaria) or virulent Listeria monocytogenes—infections that elicit robust ROS production (Postma et al., 1996Postma N.S. Mommers E.C. Eling W.M. Zuidema J. Oxidative stress in malaria; implications for prevention and therapy.Pharm. World Sci. 1996; 18: 121-129Crossref PubMed Scopus (101) Google Scholar, Serbina et al., 2003Serbina N.V. Salazar-Mather T.P. Biron C.A. Kuziel W.A. Pamer E.G. TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection.Immunity. 2003; 19: 59-70Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar). Tandem degradation was observed in the context of both murine malaria (Figure S1H) and L. monocytogenes (Figure S1I) infections, suggesting that enhanced tandem degradation occurs in multiple experimental model systems and potentially corresponds to the inflammatory status of the individual host or samples. Thus, interpretation of data can be substantially skewed when comparing cells in samples with varying inflammation and/or ROS production. We noted similar problems with frozen human peripheral blood mononuclear cell (PBMC) samples obtained from septic patients and healthy controls (Figures S1J–S1M). Although the frequency of ROS-producing leukocytes was significantly higher in septic patient samples compared to healthy controls, the amount of ROS produced, even in healthy controls samples, facilitated tandem degradation. To address whether ROS production and subsequent degradation was a result of sample cryopreservation (Paredes et al., 2018Paredes R.M. Tadaki D.K. Sooter A. Gamboni F. Sheppard F. Cryopreservation of human whole blood allows immunophenotyping by flow cytometry up to 30days after cell isolation.J. Immunol. Methods. 2018; 452: 32-38Crossref PubMed Scopus (7) Google Scholar), leukocytes were isolated from blood donation cones then split in two: the first group was left at 4°C and the other frozen in lymphocyte freeze media. After 12 h, the cells in the frozen group were thawed, and both groups were stained. The frozen cells demonstrated enhanced fluorophore degradation and ROS production (Figures S1N and S1O), and this phenomenon appeared to increase with the duration of freezing (data not shown). Therefore, the appearance of the phantom signal in frozen samples could substantially influence accurate detection and/or description of human cell subsets. Additionally, this phenomenon was exacerbated by prior infection(s) (Figure S1P), potentially due to a trained immune component, and may explain the prevalence of degradation in human samples. A strong linear relationship existed between the degradation of multiple fluorophores for a given sample (Figure S1P), suggesting that if one tandem was degrading, then they were all likely to be degrading, although some tandems showed more pronounced degradation than others (e.g., APC-Cy7 >>> PE-Cy7). Differences in susceptibility of tandems to degradation may be relevant to the likelihood of detecting a problem, such as using PerCP-Cy5.5, which is fairly resistant to degradation. Additionally, using Cy7 alternatives, such as H7, may limit problems; however, it is noteworthy that other "more stable" conjugates like APC-Fire still showed considerable degradation (Figure S1C). By understanding the factors that underlie non-uniform tandem degradation, we propose some simple adjustments to help detect, limit, or obviate this problem. (1) This problem is most easily detected by including an empty channel for a primary fluorophore that also has a tandem dye included (e.g., if staining with APC-Cy7, leave an empty APC channel). While this reduces the number of available channels, it ensures detection of fluorophore degradation. As there is a strong linear correlation between degradation of different fluorophores (i.e., PE-Cy7 to PE and APC-Cy7 to APC occurred equivalently within an individual sample; Figure S1P), degradation only needs to be monitored by a single fluorophore to determine whether a given sample may have an issue. (2) Incorporation of a reducing agent into the staining buffer. One example was the addition of BME to the staining buffer (Figure S1E). BME was chosen for its common use in complete media formulations and was utilized at a concentration consistent with that formulation. BME addition was able to limit tandem-fluorophore degradation without adversely impacting controls. Alternatively, vitamin C is another option (Le Roy et al., 2009Le Roy C. Varin-Blank N. Ajchenbaum-Cymbalista F. Letestu R. Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.Cytometry A. 2009; 75: 882-890Crossref PubMed Scopus (30) Google Scholar, Morawski et al., 2019Morawski P.A. Motley S.J. Campbell D.J. Rapid Light-Dependent Degradation of Fluorescent Dyes in Formulated Serum-Free Media.Immunohorizons. 2019; 3: 585-592Crossref PubMed Scopus (4) Google Scholar). However, addition of a reducing agent does not always fully alleviate the problem of tandem degradation due to the amount and kinetics of ROS being produced within a given sample. (3) Antibodies that recognize highly expressed targets with a clearly defined population can be used for the primary fluorophore. For example, use an APC-conjugated anti-Thy1.1 mAb, an exceptionally bright antibody with a clear positive population, when staining with APC-Cy7. The main caveat to this is that only antibodies with a distinct positive population can be used for the primary fluorophore. Further, relative protein expression (geometric mean fluorescence intensity [GMFI], mean fluorescence intensity [MFI], etc.) cannot be faithfully determined, as degrading fluorophores would non-uniformly influence this value. (4) As ROS production, as well as subsequent tandem degradation, is dependent on living cells, cells can be fixed prior to staining with tandem fluorophores and potentially further improved through the use of stabilizing fixation buffers. This, however, does pose some issues, as fixation can alter target antigens such that the antibody no longer binds (e.g., the anti-NK1.1 clone PK136). (5) Given the interrelationships between fluorophore degradation, ROS, and granulocytes, removal of granulocytes from samples prior to staining may decrease both ROS and tandem degradation. These data demonstrate how variability inherent to samples and experimental groups can strongly impact the interpretation of flow cytometry data due to tandem degradation. However, our non-exhaustive list of potential modifications should help alleviate or control for tandem degradation. Importantly, our assessment does not distinguish between the tandem truly breaking free from the fluorophore or merely disrupting the FRET capacity of the tandem. While functionally indistinguishable, future work examining this mechanism may allow for creation of better tandems or linkers for tandems to minimize the problem described here. Funding for this study was provided by NIH grants AI147064, GM113961, and GM134880 (V.P.B.); AI114543 (J.T.H. and V.P.B.); AI42767, AI85515, and AI100527 (J.T.H.); GM115462 (T.S.G.); and T32AI007511 and T32AI007485 to I.J.J. and Veterans Health Administration I01BX001324 to T.S.G. Download .pdf (1.98 MB) Help with pdf files Figure S1. Differential Tandem Fluorophore Degradation between Experimental Groups and/or Samples Obscures Interpretation of Flow Cytometric DataA) In an ideal system, tandem conjugate fluorophores would not degrade and only fluoresce in their given channel without fluorescing in the channel of their primary fluorophore. However, when tandem conjugate fluorophores degrade both the signal of the tandem conjugate fluorophore and the primary fluorophore can be present. B) P14 chimeric mice were generated by adoptive transfer of Thy1.1 P14s into Thy1.2 recipients followed by infection with LCMV-Arm. At a memory time point, mice underwent sham or CLP surgery. Day 9 post-surgery splenocytes were labeled with anti-Thy1.1-APC-efluor780. Compensation was performed for both APC-efluor780 and an empty APC channel. Representative histogram of the APC signal from APC-efluor780 labeled cells from sham (black) and CLP (red) hosts. APC GMFI [Mean: sham-179.7, CLP-624.3; standard error: sham-99.34, CLP-112.4; n: sham-3, CLP-3; p < 0.05: sham v CLP]. C) Utilizing the same experimental procedure as in panel B, cells were stained with the indicated antibody fluorophore and representative histograms of empty APC channel is shown. D) Splenocytes from naive mice were labeled with Thy1.2- APC-efluor780. During staining cells were exposed to normal FACs buffer or FACs buffer containing H2O2 (to induce oxidation). Representative histograms of empty APC channel from APC-efluor780 labeled cells exposed to either normal conditions (black) or in the presence of H2O2 (blue). E) Utilizing the same experimental design as in panel B, cells were labeled with anti-Thy1.1-APC-efluor780 in either the presence or absence of BME. Sham without BME (black), Sham with BME (gray), CLP without BME (red), and CLP with BME (blue). Representative histograms of empty APC channel for APC-efluor780 labeled cells are shown. APC GMFI [Mean: sham w/o BME-433.8, sham w/ BME-142.0, CLP w/o BME-1219, CLP w/o BME-933; standard error: sham w/o BME-71.79, sham w/ BME-2.72, CLP w/o BME-119.8, CLP w/o BME-228.2; n: sham w/o BME-5, sham w/ BME-5, CLP w/o BME-5, CLP w/ BME-5; p < 0.05: sham w/o BME v CLP w/o BME, sham w/ BME v CLP w/o BME, sham w/ BME v CLP w/ BME]. F, G) Utilizing the same experimental setup as in panel B. F) Representative gating and G) cumulative data for ROS production by splenocytes from sham (black) and CLP (red) hosts is shown. n = 5/group ∗ = p < 0.05 error bars indicate standard error. H) Mice were infected IV with Plasmodium berghei ANKA (PbANKA) radiation attenuated sporozoites (RAS) followed in some mice 2 days later by IV inoculation of PyXNL infected red blood cells to generate blood-stage infection. Parasitemia was cleared by day 35 and splenocytes from both groups were harvested on day 42. Cells were labeled with anti-Thy1.2-APC-efluor780 and -CD8a-PECy7. Representative histograms for empty APC and PE channels from RAS-immunized and blood-stage infected (red) compared to RAS-immunized controls (black) for APC-efluor780 and PE-Cy7 labeled cells. APC GMFI [Mean: PbANKA RAS-−379, PbANKA RAS+PyXNL iRBCs-1272; standard error: PbANKA RAS-14.94, PbANKA RAS+PyXNL iRBCs-421.6; n: PbANKA RAS-4, PbANKA RAS+PyXNL iRBCs-5; p < 0.05: PbANKA RAS v PbANKA RAS+PyXNL iRBCs]; PE GMFI [Mean: PbANKA RAS-23.88, PbANKA RAS+PyXNL iRBCs −163.6; standard error: PbANKA RAS-2.433, PbANKA RAS+PyXNL iRBCs-30.25; n: PbANKA RAS-4, PbANKA RAS+PyXNL iRBCs −5; p < 0.05: PbANKA RAS v PbANKA RAS+PyXNL iRBCs]. I) Mice were either left naive or infected IV with 10LD50 of virulent L.m. 10403S. 2 days post-infection, spleens were harvested and stained with anti-CD8a-PE-Cy7. Representative histograms for empty PE channel from L.m. infected (red) and naive controls (black) for PE-Cy7 labeled cells. APC GMFI [Mean: Naive-179.7, L. monocytogenes-624.3; standard error: Naive −99.34, L. monocytogenes-112.4; n: Naive −3, L. monocytogenes-4; p < 0.05: Naive v L. monocytogenes]. J) Representative histograms and K) cumulative data for ROS production by leukocytes from healthy controls (black) and septic patients (red) is shown. n = 10/group ∗ = p < 0.05 error bars indicate standard error. L, M) Frozen leukocytes from septic patients and healthy controls were thawed and stained with anti-CD45-APC-Cy7. L) Representative histograms and M) cumulative GMFI data of empty APC channel on APC-Cy7-labeled cells from healthy controls (black) and septic patients (red) APC-Cy7 control (gray). n = 10/group ∗ = p < 0.05 error bars indicate standard error. N,O) Leukocytes were isolated from blood donation cones. Samples were split and either left at 4°C or frozen in lymphocyte freeze media. 12hrs later samples were thawed and both the samples at 4°C and the frozen samples were stained with anti-CD45-APC-Cy7. N) Representative histograms of empty APC channel on APC-Cy7 labeled cells from non-frozen cells (black) and frozen cells (red) with APC-Cy7 control (gray). O) Representative histograms for ROS production by leukocytes from the same donor blood cone that were either left at 4°C (black) or frozen(red). P) Mice were either infected with LCMV-Arm or left naive. 30 days post LCMV infection mice were split into four groups: LCMV with Sham surgery (black), LCMV infected with 1x107 cf.u of attenuated (ActA deficient) L.m. (gray), Naive with CLP surgery (blue), and LCMV with CLP surgery (red). 9 days after surgery or L.m. infection splenocytes were stained with anti-CD8a-APC-Cy7 and -CD11a-PE-Cy7. GMFI of APC and PE for individual samples were graphed by each other with linear regression to determine the strength of the correlation between the factors. n: LCMV+sham-4, LCMV+L.m.-4, Naive+CLP-4, LCMV+CLP-6.

Induction of memory CD8(+) T cells is important for controlling infections such as malaria and HIV/AIDS and for cancer immunotherapy. Accurate assessment of antigen-specific (Ag-specific) CD8(+) T cells is critical for vaccine optimization and for defining correlates of protection. However, conditions for determining Ag-specific CD8(+)T cell responses ex vivo using intracellular cytokine staining (ICS) may be variable, especially in humans with complex antigens. Here, we used an attenuated whole parasite malaria vaccine model in humans and various experimental infections in mice to show that the duration of antigenic stimulation and timing of brefeldin A (BFA) addition influence the magnitude of Ag-specific and bystander T cell responses. Indeed, after immunization with an attenuated whole sporozoite malaria vaccine in humans, significantly higher numbers of IFN-gamma-producing memory CD8(+)T cells comprising Ag-specific and bystander responses were detected when the duration of Ag stimulation prior to addition of BFA was increased. Mechanistic analyses of virus-specific CD8(+)T cells in mice revealed that the increase in IFN-gamma-producing CD8(+) T cells was due to bystander activation of Ag-experienced memory CD8(+)T cells, and correlated with the proportion of Ag-experienced CD8(+)T cells in the stimulated populations. Incubation with anti-cytokine antibodies (e.g., IL-12) improved accuracy in detecting bona fide memory CD8(+)T cell responses. suggesting this as the mechanism for the bystander activation. These data have important implications for accurate assessment of immune responses generated by vaccines intended to elicit protective memory CD8(+)T cells.

Plants are continually exposed to a variety of pathogenic organisms, including bacteria, fungi and viruses. In response to these assaults, plants have developed various defense pathways to protect themselves from pathogen invasion. An understanding of the expression and regulation of genes involved in defense signaling is essential to controlling plant disease. ATL9, an Arabidopsis RING zinc finger protein, is an E3 ubiquitin ligase that is induced by chitin and involved in basal resistance to the biotrophic fungal pathogen, Golovinomyces cichoracearum (G. cichoracearum). To better understand the expression and regulation of ATL9, we studied its expression pattern and the functions of its different protein domains. Using pATL9:GUS transgenic Arabidopsis lines we found that ATL9 is expressed in numerous tissues at various developmental stages and that GUS activity was induced rapidly upon wounding. Using a GFP control protein, we showed that ATL9 is a short-lived protein within plant cells and it is degraded via the ubiquitin-proteasome pathway. ATL9 contains two transmembrane domains (TM), a RING zinc-finger domain, and a PEST domain. Using a series of deletion mutants, we found that the PEST domain and the RING domain have effects on ATL9 degradation. Further infection assays with G. cichoracearum showed that both the RING domain and the TM domains are important for ATL9's resistance phenotype. Interestingly, the PEST domain was also shown to be significant for resistance to fungal pathogens. This study demonstrates that the PEST domain is directly coupled to plant defense regulation and the importance of protein degradation in plant immunity.

Increasing use of iron oxide nanoparticles in medicine and environmental remediation has led to concerns regarding exposure of these nanoparticles to the public. However, limited studies are available to evaluate their effects on the environment, in particular on plants and food crops. Here, we investigated the effects of positive (PC) and negative (NC) charged iron oxide (Fe2O3) nanoparticles (IONPs) on the physiology and reproductive capacity of Arabidopsis thaliana at concentrations of 3 and 25 mg/L. The 3 mg/L treated plants did not show evident effects on seeding and root length. However, the 25 mg/L treatment resulted in reduced seedling (positive-20% and negative-3.6%) and root (positive-48% and negative-negligible) length. Interestingly, treatment with polyethylenimine (PEI; IONP-PC coating) also resulted in reduced root length (39%) but no change was observed with polyacrylic acid (PAA; IONP-NC coating) treatment alone. However, treatment with IONPs at 3 mg/L did lead to an almost 5% increase in aborted pollen, a 2%–6% reduction in pollen viability and up to an 11% reduction in seed yield depending on the number of treatments. Interestingly, the treated plants did not show any observable phenotypic changes in overall size or general plant structure, indicating that environmental nanoparticle contamination could go dangerously unnoticed.