Potato Non-Specific Lipid Transfer Protein StnsLTPI.33 Is Associated with the Production of Reactive Oxygen Species, Plant Growth, and Susceptibility to Alternaria solani.

Plant non-specific lipid transfer proteins (nsLTPs) are small proteins capable of transferring phospholipids between membranes and binding non-specifically fatty acids in vitro. They constitute large gene families in plants, e.g., 83 in potato (Solanum tuberosum). Despite their recognition decades ago, very few have been functionally characterized. Here, we set out to better understand the function of one of the potato members, StnsLTPI.33. Using quantitative polymerase chain reaction, we show that StnsLTPI.33 is expressed throughout the potato plant, but at relatively higher levels in roots and leaves compared to petals, anthers, and the ovary. We also show that ectopically-expressed StnsLTPI.33 fused to green fluorescent protein colocalized with an apoplastic marker in Nicotiana benthamiana leaves, indicating that StnsLTPI.33 is targeted to the apoplast. Constitutive overexpression of the StnsLTPI.33 gene in potato led to increased levels of superoxide anions and reduced plant growth, particularly under salt stress conditions, and enhanced susceptibility to Alternaria solani. In addition, StnsLTPI.33-overexpressing plants had a depleted leaf pool of pipecolic acid, threonic acid, and glycine, while they accumulated putrescine. To our knowledge, this is the first report of an nsLTP that is associated with enhanced susceptibility to a pathogen in potato.

Ill Communication: Host Metabolites as Virulence-Regulating Signals for Plant-Pathogenic Bacteria.

Plant bacterial pathogens rely on host-derived signals to coordinate the deployment of virulence factors required for infection. In this review, I describe how diverse plant-pathogenic bacteria detect and respond to plant-derived metabolic signals for the purpose of virulence gene regulation. I highlight examples of how pathogens perceive host metabolites through membrane-localized receptors as well as intracellular response mechanisms. Furthermore, I describe how individual strains may coordinate their virulence using multiple distinct host metabolic signals, and how plant signals may positively or negatively regulate virulence responses. I also describe how plant defenses may interfere with the perception of host metabolites as a means to dampen pathogen virulence. The emerging picture is that recognition of host metabolic signals for the purpose of virulence gene regulation represents an important primary layer of interaction between pathogenic bacteria and host plants that shapes infection outcomes.

Plant-exuded chemical signals induce surface attachment of the bacterial pathogen Pseudomonas syringae.

Many plant pathogenic bacteria suppress host defenses by secreting small molecule toxins or immune-suppressing proteins into host cells, processes that likely require close physical contact between pathogen and host. Yet, in most cases, little is known about whether phytopathogenic bacteria physically attach to host surfaces during infection. Here we report that Pseudomonas syringae pv. tomato strain DC3000, a Gram-negative bacterial pathogen of tomato and Arabidopsis, attaches to polystyrene and glass surfaces in response to chemical signals exuded from Arabidopsis seedlings and tomato leaves. We characterized the molecular nature of these attachment-inducing signals and discovered that multiple hydrophilic metabolites found in plant exudates, including citric acid, glutamic acid, and aspartic acid, are potent inducers of surface attachment. These same compounds were previously identified as inducers of P. syringae genes encoding a type III secretion system (T3SS), indicating that both attachment and T3SS deployment are induced by the same plant signals. To test if surface attachment and T3SS are regulated by the same signaling pathways, we assessed the attachment phenotypes of several previously characterized DC3000 mutants, and found that the T3SS master regulator HrpL was partially required for maximal levels of surface attachment, whereas the response regulator GacA, a negative regulator of T3SS, negatively regulated DC3000 surface attachment. Together, our data indicate that T3SS deployment and surface attachment by P. syringae may be co-regulated by the same host signals during infection, possibly to ensure close contact necessary to facilitate delivery of T3SS effectors into host cells.

Chronic blue light leads to accelerated aging in Drosophila by impairing energy metabolism and neurotransmitter levels.

Blue light (BL) is becoming increasingly prevalent in artificial illumination, raising concerns about its potential health hazard to humans. In fact, there is evidence suggesting that acute BL exposure may lead to oxidative stress and death of retinal cells specialized for photoreception. On the other hand, recent studies in Drosophila melanogaster demonstrated that chronic BL exposure across lifespan leads to accelerated aging manifested in reduced lifespan and brain neurodegeneration even in flies with genetically ablated eyes, suggesting that BL can damage cells and tissues not specialized for light perception. At the physiological level, BL exposure impairs mitochondria function in flies, but the metabolic underpinnings of these effects have not been studied. Here, we investigated effects of chronic BL on metabolic pathways in heads of eyes absent (eya 2 ) mutant flies in order to focus on extra-retinal tissues. We compared metabolomic profiles in flies kept for 10 or 14 days in constant BL or constant darkness, using LC-MS and GC-MS. Data analysis revealed significant alterations in the levels of several metabolites suggesting that critical cellular pathways are impacted in BL-exposed flies. In particular, dramatic metabolic rearrangements are observed in heads of flies kept in BL for 14 days, including highly elevated levels of succinate but reduced levels of pyruvate and citrate, suggesting impairments in energy production. These flies also show onset of neurodegeneration and our analysis detected significantly reduced levels of several neurotransmitters including glutamate and Gamma-aminobutyric acid (GABA), suggesting that BL disrupts brain homeostasis. Taken together, these data provide novel insights into the mechanisms by which BL interferes with vital metabolic pathways that are conserved between fly and human cells.

Regulation of the Pseudomonas syringae Type III Secretion System by Host Environment Signals.

Pseudomonas syringae are Gram-negative, plant pathogenic bacteria that use a type III secretion system (T3SS) to disarm host immune responses and promote bacterial growth within plant tissues. Despite the critical role for type III secretion in promoting virulence, T3SS-encoding genes are not constitutively expressed by P. syringae and must instead be induced during infection. While it has been known for many years that culturing P. syringae in synthetic minimal media can induce the T3SS, relatively little is known about host signals that regulate the deployment of the T3SS during infection. The recent identification of specific plant-derived amino acids and organic acids that induce T3SS-inducing genes in P. syringae has provided new insights into host sensing mechanisms. This review summarizes current knowledge of the regulatory machinery governing T3SS deployment in P. syringae, including master regulators HrpRS and HrpL encoded within the T3SS pathogenicity island, and the environmental factors that modulate the abundance and/or activity of these key regulators. We highlight putative receptors and regulatory networks involved in linking the perception of host signals to the regulation of the core HrpRS-HrpL pathway. Positive and negative regulation of T3SS deployment is also discussed within the context of P. syringae infection, where contributions from distinct host signals and regulatory networks likely enable the fine-tuning of T3SS deployment within host tissues. Last, we propose future research directions necessary to construct a comprehensive model that (a) links the perception of host metabolite signals to T3SS deployment and (b) places these host-pathogen signaling events in the overall context of P. syringae infection.

Arabidopsis group C Raf-like protein kinases negatively regulate abscisic acid signaling and are direct substrates of SnRK2.

The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur.

StPIP1, a PAMP-induced peptide in potato, elicits plant defenses and is associated with disease symptom severity in a compatible interaction with Potato virus Y.

The role of small secreted peptides in plant defense responses to viruses has seldom been investigated. Here, we report a role for potato (Solanum tuberosum) PIP1, a gene predicted to encode a member of the pathogen-associated molecular pattern (PAMP)-induced peptide (PIP) family, in the response of potato to Potato virus Y (PVY) infection. We show that exogenous application of synthetic StPIP1 to potato leaves and nodes increased the production of reactive oxygen species and the expression of plant defense-related genes, revealing that StPIP1 triggers early defense responses. In support of this hypothesis, transgenic potato plants that constitutively overexpress StPIP1 had higher levels of leaf callose deposition and, based on measurements of viral RNA titers, were less susceptible to infection by a compatible PVY strain. Interestingly, systemic infection of StPIP1-overexpressing lines with PVY resulted in clear rugose mosaic symptoms that were absent or very mild in infected non-transgenic plants. A transcriptomics analysis revealed that marker genes associated with both pattern-triggered immunity and effector-triggered immunity were induced in infected StPIP1 overexpressors but not in non-transgenic plants. Together, our results reveal a role for StPIP1 in eliciting plant defense responses and in regulating plant antiviral immunity.

Ancient co-option of an amino acid ABC transporter locus in Pseudomonas syringae for host signal-dependent virulence gene regulation.

Pathogenic bacteria frequently acquire virulence traits via horizontal gene transfer, yet additional evolutionary innovations may be necessary to integrate newly acquired genes into existing regulatory pathways. The plant bacterial pathogen Pseudomonas syringae relies on a horizontally acquired type III secretion system (T3SS) to cause disease. T3SS-encoding genes are induced by plant-derived metabolites, yet how this regulation occurs, and how it evolved, is poorly understood. Here we report that the two-component system AauS-AauR and substrate-binding protein AatJ, proteins encoded by an acidic amino acid-transport (aat) and -utilization (aau) locus in P. syringae, directly regulate T3SS-encoding genes in response to host aspartate and glutamate signals. Mutants of P. syringae strain DC3000 lacking aauS, aauR or aatJ expressed lower levels of T3SS genes in response to aspartate and glutamate, and had decreased T3SS deployment and virulence during infection of Arabidopsis. We identified an AauR-binding motif (Rbm) upstream of genes encoding T3SS regulators HrpR and HrpS, and demonstrated that this Rbm is required for maximal T3SS deployment and virulence of DC3000. The Rbm upstream of hrpRS is conserved in all P. syringae strains with a canonical T3SS, suggesting AauR regulation of hrpRS is ancient. Consistent with a model of conserved function, an aauR deletion mutant of P. syringae strain B728a, a bean pathogen, had decreased T3SS expression and growth in host plants. Together, our data suggest that, upon acquisition of T3SS-encoding genes, a strain ancestral to P. syringae co-opted an existing AatJ-AauS-AauR pathway to regulate T3SS deployment in response to specific host metabolite signals.

EPSIN1 Modulates the Plasma Membrane Abundance of FLAGELLIN SENSING2 for Effective Immune Responses.

The plasma membrane (PM) provides a critical interface between plant cells and their environment to control cellular responses. To perceive the bacterial flagellin peptide flg22 for effective defense signaling, the immune receptor FLAGELLIN SENSING2 (FLS2) needs to be at its site of function, the PM, in the correct abundance. However, the intracellular machinery that controls PM accumulation of FLS2 remains largely undefined. The Arabidopsis (Arabidopsis thaliana) clathrin adaptor EPSIN1 (EPS1) is implicated in clathrin-coated vesicle formation at the trans-Golgi network (TGN), likely aiding the transport of cargo proteins from the TGN for proper location; but EPS1's impact on physiological responses remains elusive. Here, we identify EPS1 as a positive regulator of flg22 signaling and pattern-triggered immunity against Pseudomonas syringae pv tomato DC3000. We provide evidence that EPS1 contributes to modulating the PM abundance of defense proteins for effective immune signaling because in eps1, impaired flg22 signaling correlated with reduced PM accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). The eps1 mutant also exhibited reduced responses to the pathogen/damage-associated molecular patterns elf26 and AtPep1, which are perceived by the coreceptor BAK1 and cognate PM receptors. Furthermore, quantitative proteomics of enriched PM fractions revealed that EPS1 was required for proper PM abundance of a discrete subset of proteins with different cellular functions. In conclusion, our study expands the limited understanding of the physiological roles of EPSIN family members in plants and provides novel insight into the TGN-associated clathrin-coated vesicle trafficking machinery that impacts plant PM-derived defense processes.

A revised model for the role of GacS/GacA in regulating type III secretion by Pseudomonas syringae pv. tomato DC3000.

GacS/GacA is a conserved two-component system that functions as a master regulator of virulence-associated traits in many bacterial pathogens, including Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/GacA-regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogen Pseudomonas aeruginosa, GacS/GacA negatively regulates the expression of type III secretion system (T3SS)-encoding genes. However, in the plant pathogenic bacterium Pseudomonas syringae, strain-to-strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we re-evaluated the function of GacA in P. syringae pv. tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence inside Arabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface-inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulating P. syringae virulence.

A DeoR-Type Transcription Regulator Is Required for Sugar-Induced Expression of Type III Secretion-Encoding Genes in Pseudomonas syringae pv. tomato DC3000.

The type III secretion system (T3SS) of plant-pathogenic Pseudomonas syringae is essential for virulence. Genes encoding the T3SS are not constitutively expressed and must be induced upon infection. Plant-derived metabolites, including sugars such as fructose and sucrose, are inducers of T3SS-encoding genes, yet the molecular mechanisms underlying perception of these host signals by P. syringae are unknown. Here, we report that sugar-induced expression of type III secretion A (setA), predicted to encode a DeoR-type transcription factor, is required for maximal sugar-induced expression of T3SS-associated genes in P. syringae DC3000. From a Tn5 transposon mutagenesis screen, we identified two independent mutants with insertions in setA. When both setA::Tn5 mutants were cultured in minimal medium containing fructose, genes encoding the T3SS master regulator HrpL and effector AvrRpm1 were expressed at lower levels relative to that of a wild-type strain. Decreased hrpL and avrRpm1 expression also occurred in a setA::Tn5 mutant in response to glucose, sucrose, galactose, and mannitol, demonstrating that setA is genetically required for T3SS induction by many different sugars. Expression of upstream regulators hrpR/S and rpoN was not altered in setA::Tn5, indicating that SetA positively regulates hrpL expression independently of increased transcription of these genes. In addition to decreased response to defined sugar signals, a setA::Tn5 mutant had decreased T3SS deployment during infection and was compromised in its ability to grow in planta and cause disease. These data suggest that SetA is necessary for P. syringae to effectively respond to T3SS-inducing sugar signals encountered during infection.

Re-evaluation of a Tn5::gacA mutant of Pseudomonas syringae pv. tomato DC3000 uncovers roles for uvrC and anmK in promoting virulence.

Pseudomonas syringae is a taxon of plant pathogenic bacteria that can colonize and proliferate within the interior space of leaf tissue. This process requires P. syringae to rapidly upregulate the production of virulence factors including a type III secretion system (T3SS) that suppress host defenses. GacS/A is a two-component system that regulates virulence of many plant and animal pathogenic bacteria including P. syringae. We recently investigated the virulence defect of strain AC811, a Tn5::gacA mutant of P. syringae pv. tomato DC3000 that is less virulent on Arabidopsis. We discovered that decreased virulence of AC811 is not caused by loss of GacA function. Here, we report the molecular basis of the virulence defect of AC811. We show that AC811 possesses a nonsense mutation in anmK, a gene predicted to encode a 1,6-anhydromuramic acid kinase involved in cell wall recycling. Expression of a wild-type allele of anmK partially increased growth of AC811 in Arabidopsis leaves. In addition to the defective anmK allele, we also show that the Tn5 insertion in gacA exerts a polar effect on uvrC, a downstream gene encoding a regulator of DNA damage repair. Expression of the wild-type anmK allele together with increased expression of uvrC fully restored the virulence of AC811 during infection of Arabidopsis. These results demonstrate that defects in anmK and uvrC are together sufficient to account for the decreased virulence of AC811, and suggest caution is warranted in assigning phenotypes to GacA function based on insertional mutagenesis of the gacA-uvrC locus.

Isolation and Characterization of Plant Metabolite Signals that Induce Type III Secretion by the Plant Pathogen Pseudomonas syringae.

Pseudomonas syringae is a bacterium that can cause disease on a wide range of plant species including important agricultural crops. A primary virulence mechanism used by P. syringae to infect host plants is the type III secretion system (T3SS), a syringe-like structure that delivers defense-suppressing proteins directly into plant cells. Genes encoding the T3SS are not transcribed in P. syringae prior to contact with a potential host plant and must be expressed during initial stages of infection. Specific organic and amino acids exuded by plants were recently identified as signals that can induce expression of T3SS-associated genes. Here we describe a technique to produce exudates from intact Arabidopsis seedlings and evaluate the exudates for the presence of these bioactive metabolites. We provide procedures for exudate production as well as downstream assays to assess T3SS gene expression using a GFP transcriptional reporter. We also describe methods for preparing high-quality protein and RNA from exudate-treated bacteria to directly assess changes in mRNA and protein abundance. These methods could be used to investigate mechanisms regulating P. syringae perception of plant metabolites as well as the release of these substances by the plant, and more generally to investigate host signals perceived by other phytopathogens.

Development of a Pseudomonas syringae-Arabidopsis Suspension Cell Infection System for Investigating Host Metabolite-Dependent Regulation of Type III Secretion and Pattern-Triggered Immunity.

The importance of pattern-triggered immunity (PTI) in plant defense has been clearly established through genetic studies of mutants lacking functional pattern recognition receptors (PRRs) and signaling components downstream of PRR activation. Despite extensive knowledge of PRR-mediated signaling responses to pathogen-associated molecular patterns (PAMPs), little is known about which of these responses, if any, are directly responsible for limiting bacterial growth. In this work, we established a protocol for coculturing the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and Arabidopsis suspension cells. The system closely mirrors infection processes that occur in leaves, with bacteria relying on the type III secretion system (T3SS) for maximal growth and PAMP-induced plant defenses effectively limiting bacterial growth. To demonstrate the utility of this system, we investigated the molecular basis of PAMP-induced growth inhibition and discovered that T3SS-associated genes are inhibited when DC3000 is cocultured with PAMP-treated plant suspension cells. To determine the underlying mechanism of decreased T3SS gene expression, we performed metabolomics and biochemical analyses of suspension cell exudates and identified 14 metabolites that significantly increased or decreased following PAMP treatment. Citric acid, a known inducer of T3SS gene expression in DC3000, was among several organic acids decreased in exudates from PAMP-treated plant cells. Exogenous addition of citric acid increased T3SS gene expression and partially recovered growth of DC3000 in the presence of PAMP-treated cells, indicating that a portion of PAMP-induced defense in this system is decreased extracellular release of this metabolite. We envision that the well-defined infection conditions of this coculture system will be valuable for quantitative studies of type III effector delivery by P. syringae. Furthermore, this system provides a unique 'top-down' approach to unravel the molecular basis of PTI against P. syringae.

Phosphorylation of Arabidopsis MAP Kinase Phosphatase 1 (MKP1) Is Required for PAMP Responses and Resistance against Bacteria.

Plants perceive potential pathogens via the recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors, which initiates a series of intracellular responses that ultimately limit bacterial growth. PAMP responses include changes in intracellular protein phosphorylation, including the activation of mitogen-activated protein kinase (MAPK) cascades. MAP kinase phosphatases (MKPs), such as Arabidopsis (Arabidopsis thaliana) MKP1, are important negative regulators of MAPKs and play a crucial role in controlling the intensity and duration of MAPK activation during innate immune signaling. As such, the mkp1 mutant lacking MKP1 displays enhanced PAMP responses and resistance against the virulent bacterium Pseudomonas syringae pv tomato DC3000. Previous in vitro studies showed that MKP1 can be phosphorylated and activated by MPK6, suggesting that phosphorylation may be an important mechanism for regulating MKP1. We found that MKP1 was phosphorylated during PAMP elicitation and that phosphorylation stabilized the protein, resulting in protein accumulation after elicitation. MKP1 also can be stabilized by the proteasome inhibitor MG132, suggesting that MKP1 is constitutively degraded through the proteasome in the resting state. In addition, we investigated the role of MKP1 posttranslational regulation in plant defense by testing whether phenotypes of the mkp1 Arabidopsis mutant could be complemented by expressing phosphorylation site mutations of MKP1. The phosphorylation of MKP1 was found to be required for some, but not all, of MKP1's functions in PAMP responses and defense against bacteria. Together, our results provide insight into the roles of phosphorylation in the regulation of MKP1 during PAMP signaling and resistance to bacteria.

Genetic dissection of Arabidopsis MAP kinase phosphatase 1-dependent PAMP-induced transcriptional responses.

Plant immunity is initiated by extracellular detection of pathogen-associated molecular patterns (PAMPs) through surface-localized pattern recognition receptors (PRRs). PRR activation induces many responses including the activation of mitogen-activated protein kinases (MAPKs) that ultimately limit bacterial growth. Previous work identified Arabidopsis MAP kinase phosphatase 1 (MKP1) as a negative regulator of signaling pathways required for some, but not all, of PAMP-initiated responses. Specifically, loss of MAPK MPK6 in an mkp1 background suppressed a subset of the mkp1-dependent biological phenotypes, indicating the requirement for MPK6 in MKP1-dependent signaling. To further genetically separate the outputs of PAMP-responsive signaling pathways, we performed a transcriptome analysis in Arabidopsis wild type, mkp1 and mkp1 mpk6 seedlings treated with the bacterially derived PAMP elf26 for 0, 30, and 90 min. Using differential genetic and temporal clustering analyses between and within genotypes, we identified and separated 6963 elf26-responsive transcripts based on both genetic requirements of MKP1 (with or without a requirement for MPK6) and temporal transcriptional accumulation patterns, and some of these novel response markers were validated by qRT-PCR over a more extended time course. Taken together, our transcriptome analysis provides novel information for delineating PAMP signaling pathways.

Plasma membrane proteomics in the maize primary root growth zone: novel insights into root growth adaptation to water stress.

Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress.

Cell wall protection by the Candida albicans class I chitin synthases.

Candida albicans has four chitin synthases from three different enzyme classes which deposit chitin in the cell wall, including at the polarized tips of growing buds and hyphae, and sites of septation. The two class I enzymes, Chs2 and Chs8, are responsible for most of the measurable chitin synthase activity in vitro, but their precise biological functions in vivo remain obscure. In this work, detailed phenotypic analyses of a chs2Δchs8Δ mutant have shown that C. albicans class I chitin synthases promote cell integrity during early polarized growth in yeast and hyphal cells. This was supported by live cell imaging of YFP-tagged versions of the class I chitin synthases which revealed that Chs2-YFP was localized at sites of polarized growth. Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed. Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation. Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses.