Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process.

Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry. This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and colocalize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome dataset will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.

Dynamic localization of a helper NLR at the plant-pathogen interface underpins pathogen recognition.

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet, the subcellular localization of NLRs pre- and postactivation during pathogen infection remains poorly understood. Here, we show that NRC4, from the "NRC" solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extrahaustorial membrane (EHM), presumably to mediate response to perihaustorial effectors that are recognized by NRC4-dependent sensor NLRs. However, not all NLRs accumulate at the EHM, as the closely related helper NRC2 and the distantly related ZAR1 did not accumulate at the EHM. NRC4 required an intact N-terminal coiled-coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that postactivation, NRC4 may undergo a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection, however, NRC4 forms puncta mainly at the EHM and, to a lesser extent, at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses.

Phytophthora infestans RxLR Effector PITG06478 Hijacks 14-3-3 to Suppress PMA Activity Leading to Necrotrophic Cell Death.

Pathogens often induce cell death for their successful proliferation in the host plant. Plasma membrane H+-ATPases (PMAs) are targeted by either pathogens or plant immune receptors in immune response regulation. Although PMAs play pivotal roles in host cell death, the molecular mechanism of effector-mediated regulation of PMA activity has not been described. Here, we report that the Phytophthora infestans RxLR effector PITG06478 can induce cell death in Nicotiana benthamiana but the induced cell death is inhibited by fusicoccin (FC), an irreversible PMA activator. PITG06478, which is localized at the plasma membrane, is not directly associated with the PMA but is associated with Nb14-3-3s, a PMA activator. Immunoblot analyses revealed that the interaction between PITG06478 and Nb14-3-3s was disrupted by FC. PMA activity in PITG06478-expressing plants was eventually inhibited, and cell death likely occurred because the 14-3-3 protein was hijacked. Our results further confirm the significance of PMA activity in host cell death and provide new insight into how pathogens utilize essential host components to sustain their life cycle. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Potato protein tyrosine phosphatase StPTP1a is activated by StMKK1 to negatively regulate plant immunity.

Phytophthora infestans causes severe losses in potato production. The MAPK kinase StMKK1 was previously found to negatively regulate potato immunity to P. infestans. Our results showed that StMKK1 interacts with a protein tyrosine phosphatase, referred to as StPTP1a, and StMKK1 directly phosphorylates StPTP1a at residues Ser-99, Tyr-223 and Thr-290. StPTP1a is a functional phosphatase and the phosphorylation of StPTP1a at these three residues enhances its stability and catalytic activity. StPTP1a negatively regulates potato immunity and represses SA-related gene expression. Furthermore, StPTP1a interacts with, and dephosphorylates, the StMKK1 downstream signalling targets StMPK4 and -7 at their Tyr-203 residue resulting in the repression of salicylic acid (SA)-related immunity. Silencing of NbPTP1a + NbMPK4 or NbPTP1a + NbMPK7 abolished the plant immunity to P. infestans caused by NbPTP1a silencing, indicating that PTP1a functions upstream of NbMPK4 and NbMPK7. StMKK1 requires StPTP1a to negatively regulate SA-related immunity and StPTP1a is phosphorylated and stabilized during immune activation to promote the de-phosphorylation of StMPK4 and -7. Our results reveal that potato StMKK1 activates and stabilizes the tyrosine phosphatase StPTP1a that in its turn de-phosphorylates StMPK4 and -7, thereby repressing plant SA-related immunity.

Nucleotide-binding leucine-rich repeat network underlies nonhost resistance of pepper against the Irish potato famine pathogen Phytophthora infestans.

Nonhost resistance (NHR) is a robust plant immune response against non-adapted pathogens. A number of nucleotide-binding leucine-rich repeat (NLR) proteins that recognize non-adapted pathogens have been identified, although the underlying molecular mechanisms driving robustness of NHR are still unknown. Here, we screened 57 effectors of the potato late blight pathogen Phytophthora infestans in nonhost pepper (Capsicum annuum) to identify avirulence effector candidates. Selected effectors were tested against 436 genome-wide cloned pepper NLRs, and we identified multiple functional NLRs that recognize P. infestans effectors and confer disease resistance in the Nicotiana benthamiana as a surrogate system. The identified NLRs were homologous to known NLRs derived from wild potatoes that recognize P. infestans effectors such as Avr2, Avrblb1, Avrblb2, and Avrvnt1. The identified CaRpi-blb2 is a homologue of Rpi-blb2, recognizes Avrblb2 family effectors, exhibits feature of lineage-specifically evolved gene in microsynteny and phylogenetic analyses, and requires pepper-specific NRC (NLR required for cell death)-type helper NLR for proper function. Moreover, CaRpi-blb2-mediated hypersensitive response and blight resistance were more tolerant to suppression by the PITG_15 278 than those mediated by Rpi-blb2. Combined results indicate that pepper has stacked multiple NLRs recognizing effectors of non-adapted P. infestans, and these NLRs could be more tolerant to pathogen-mediated immune suppression than NLRs derived from the host plants. Our study suggests that NLRs derived from nonhost plants have potential as untapped resources to develop crops with durable resistance against fast-evolving pathogens by stacking the network of nonhost NLRs into susceptible host plants.

An atypical Phytophthora sojae RxLR effector manipulates host vesicle trafficking to promote infection.

In plants, the apoplast is a critical battlefield for plant-microbe interactions. Plants secrete defense-related proteins into the apoplast to ward off the invasion of pathogens. How microbial pathogens overcome plant apoplastic immunity remains largely unknown. In this study, we reported that an atypical RxLR effector PsAvh181 secreted by Phytophthora sojae, inhibits the secretion of plant defense-related apoplastic proteins. PsAvh181 localizes to plant plasma membrane and essential for P. sojae infection. By co-immunoprecipitation assay followed by liquid chromatography-tandem mass spectrometry analyses, we identified the soybean GmSNAP-1 as a candidate host target of PsAvh181. GmSNAP-1 encodes a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein, which associates with GmNSF of the SNARE complex functioning in vesicle trafficking. PsAvh181 binds to GmSNAP-1 in vivo and in vitro. PsAvh181 interferes with the interaction between GmSNAP-1 and GmNSF, and blocks the secretion of apoplastic defense-related proteins, such as pathogenesis-related protein PR-1 and apoplastic proteases. Taken together, these data show that an atypical P. sojae RxLR effector suppresses host apoplastic immunity by manipulating the host SNARE complex to interfere with host vesicle trafficking pathway.

Laminarin-triggered defence responses are geographically dependent in natural populations of Solanum chilense.

Natural plant populations are polymorphic and show intraspecific variation in resistance properties against pathogens. The activation of the underlying defence responses can depend on variation in perception of pathogen-associated molecular patterns or elicitors. To dissect such variation, we evaluated the responses induced by laminarin (a glucan, representing an elicitor from oomycetes) in the wild tomato species Solanum chilense and correlated this to observed infection frequencies of Phytophthora infestans. We measured reactive oxygen species burst and levels of diverse phytohormones upon elicitation in 83 plants originating from nine populations. We found high diversity in basal and elicitor-induced levels of each component. Further we generated linear models to explain the observed infection frequency of P. infestans. The effect of individual components differed dependent on the geographical origin of the plants. We found that the resistance in the southern coastal region, but not in the other regions, was directly correlated to ethylene responses and confirmed this positive correlation using ethylene inhibition assays. Our findings reveal high diversity in the strength of defence responses within a species and the involvement of different components with a quantitatively different contribution of individual components to resistance in geographically separated populations of a wild plant species.

Mesoporous Silica Nanoparticles Induce Intracellular Peroxidation Damage of Phytophthora infestans: A New Type of Green Fungicide for Late Blight Control.

Nanopesticides are considered to be a promising alternative strategy for enhancing bioactivity and delaying the development of pathogen resistance to pesticides. Here, a new type of nanosilica fungicide was proposed and demonstrated to control late blight by inducing intracellular peroxidation damage to Phytophthora infestans, the pathogen associated with potato late blight. Results indicated that the structural features of different silica nanoparticles were largely responsible for their antimicrobial activities. Mesoporous silica nanoparticles (MSNs) exhibited the highest antimicrobial activity with a 98.02% inhibition rate of P. infestans, causing oxidative stress responses and cell structure damage in P. infestans. For the first time, MSNs were found to selectively induce spontaneous excess production of intracellular reactive oxygen species in pathogenic cells, including hydroxyl radicals (•OH), superoxide radicals (•O2-), and singlet oxygen (1O2), leading to peroxidation damage in P. infestans. The effectiveness of MSNs was further tested in the pot experiments as well as leaf and tuber infection, and successful control of potato late blight was achieved with high plant compatibility and safety. This work provides new insights into the antimicrobial mechanism of nanosilica and highlights the use of nanoparticles for controlling late blight with green and highly efficient nanofungicides.

Comparison of the Distinct, Host-Specific Response of Three Solanaceae Hosts Induced by Phytophthora infestans.

Three Solanaceae hosts (TSHs), S. tuberosum, N. benthamiana and S. lycopersicum, represent the three major phylogenetic clades of Solanaceae plants infected by Phytophthora infestans, which causes late blight, one of the most devastating diseases seriously affecting crop production. However, details regarding how different Solanaceae hosts respond to P. infestans are lacking. Here, we conducted RNA-seq to analyze the transcriptomic data from the TSHs at 12 and 24 h post P. infestans inoculation to capture early expression effects. Macroscopic and microscopic observations showed faster infection processes in S. tuberosum than in N. benthamiana and S. lycopersicum under the same conditions. Analysis of the number of genes and their level of expression indicated that distinct response models were adopted by the TSHs in response to P. infestans. The host-specific infection process led to overlapping but distinct in GO terms and KEGG pathways enriched for differentially expressed genes; many were tightly linked to the immune response in the TSHs. S. tuberosum showed the fastest response and strongest accumulation of reactive oxygen species compared with N. benthamiana and S. lycopersicum, which also had similarities and differences in hormone regulation. Collectively, our study provides an important reference for a better understanding of late blight response mechanisms of different Solanaceae host interactions.

LncRNA33732-respiratory burst oxidase module associated with WRKY1 in tomato- Phytophthora infestans interactions.

Our previous studies indicated that tomato WRKY1 transcription factor acts as a positive regulator during tomato resistance to Phytophthora infestans. However, the molecular mechanism of WRKY1-mediated resistance regulation remains unclear. Here, we used a comparative transcriptome analysis between wild-type and WRKY1-overexpressing tomato plants to identify differentially expressed genes (DEGs) and long non-coding RNAs (DELs), and we examined long non-coding RNA (lncRNA)-gene networks. The promoter sequences of the upregulated DEGs and DELs were analyzed. Among 1073 DEGs and 199 DELs, 1 kb 5'-upstream regions of 59 DEGs and 22 DELs contain the W-box, the target sequence of the WRKY1. The results of promoter-ß-glucuronidase (GUS) fusion and yeast one-hybrid assay showed that lncRNA33732 was activated by WRKY1 through sequence-specific interactions with the W-box element in its promoter. The overexpression and silencing analysis of lncRNA33732 in tomato showed that lncRNA33732 acts as a positive regulator and enhanced tomato resistance to P. infestans by induction of the expression of respiratory burst oxidase (RBOH) and increase in the accumulation of H2 O2 . When the expression of RBOH gene was inhibited in tomato plants, H2 O2 accumulation decreased and resistance were impaired. These findings suggest that lncRNA33732 activated by WRKY1 induces RBOH expression to increase H2 O2 accumulation in early defense reaction of tomato to P. infestans attack. Our results provide insights into the WRKY1-lncRNA33732-RBOH module involved in the regulation of H2 O2 accumulation and resistance to P. infestans, as well as provide candidates to enhance broad-spectrum resistance to pathogens in tomato.

Global transcriptome analyses reveal the molecular signatures in the early response of potato (Solanum tuberosum L.) to Phytophthora infestans, Ralstonia solanacearum, and Potato virus Y infection.

MAIN CONCLUSION: Specific and common genes including transcription factors, resistance genes and pathways were significantly induced in potato by Phytophthora infestans, Ralstonia solanacearum, and Potato virus Y infection. The three major pathogens, namely, Phytophthora infestans, Ralstonia solanacearum, and Potato virus Y, can cause late blight, bacterial wilt, and necrotic ringspot, respectively, and thus severely reduce the yield and quality of potatoes (Solanum tuberosum L.). This study was the first to systematically analyze the relationship between transcriptome alterations in potato infected by these pathogens at the early stages. A total of 75,500 unigenes were identified, and 44,008 were annotated into 5 databases, namely, non-redundant (NR), Swiss-Prot protein, clusters of orthologous groups for eukaryotic complete genomes (KOG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A total of 6945 resistance genes and 11,878 transcription factors (TFs) were identified from all transcriptome data. Differential expression analysis revealed that 13,032 (9490 specifics), 9877 (6423 specifics), and 6661 (4144 specifics) differentially expressed genes (DEGs) were generated from comparisons of the P. infestans/control (Pi vs. Pi-CK), R. solanacearum/control (Rs vs. Rs-CK), and PVY/control (PVY vs. PVY-CK) treatments, respectively. The specific DEGs from the 3 comparisons were assigned to 13 common pathways, such as biosynthesis of amino acids, plant hormone signal transduction, carbon metabolism, and starch and sucrose metabolism. Weighted Gene Co-Expression Network Analysis (WGCNA) identified many hub unigenes, of which several unigenes were reported to regulate plant immune responses, such as FLAGELLIN-SENSITIVE 2 and chitinases. The present study provide crucial systems-level insights into the relationship between transcriptome changes in potato infected with the three pathogens. Moreover, this study presents a theoretical basis for breeding broad-spectrum and specific pathogen-resistant cultivars.

Phytophthora infestans Sporangia Produced in Culture and on Tomato Leaflet Lesions Show Marked Differences in Indirect Germination Rates, Aggressiveness, and Global Transcription Profiles.

Sporangia of Phytophthora infestans from pure cultures on agar plates are typically used in lab studies, whereas sporangia from leaflet lesions drive natural infections and epidemics. Multiple assays were performed to determine if sporangia from these two sources are equivalent. Sporangia from plate cultures showed much lower rates of indirect germination and produced much less disease in field and moist-chamber tests. This difference in aggressiveness was observed whether the sporangia had been previously incubated at 4°C (to induce indirect germination) or at 21°C (to prevent indirect germination). Furthermore, lesions caused by sporangia from plates produced much less sporulation. RNA-Seq analysis revealed that thousands of the >17,000 P. infestans genes with a RPKM (reads per kilobase of exon model per million mapped reads) >1 were differentially expressed in sporangia obtained from plate cultures of two independent field isolates compared with sporangia of those isolates from leaflet lesions. Among the significant differentially expressed genes (DEGs), putative RxLR effectors were overrepresented, with almost half of the 355 effectors with RPKM >1 being up- or downregulated. DEGs of both isolates include nine flagellar-associated genes, and all were down-regulated in plate sporangia. Ten elicitin genes were also detected as DEGs in both isolates, and nine (including INF1) were up-regulated in plate sporangia. These results corroborate previous observations that sporangia produced from plates and leaflets sometimes yield different experimental results and suggest hypotheses for potential mechanisms. We caution that use of plate sporangia in assays may not always produce results reflective of natural infections and epidemics.

Transcriptional induction of capsidiol synthesis genes by wounding can promote pathogen signal-induced capsidiol synthesis.

BACKGROUND: Plants are exposed to various forms of environmental stress. Penetration by pathogens is one of the most serious environmental insults. Wounding caused by tissue damage or herbivory also affects the growth and reproduction of plants. Moreover, wounding disrupts physical barriers present at the plant surface and increases the risk of pathogen invasion. Plants cope with environmental stress by inducing a variety of responses. These stress responses must be tightly controlled, because their unnecessary induction is detrimental to plant growth. In tobacco, WIPK and SIPK, two wound-responsive mitogen-activated protein kinases, have been shown to play important roles in regulating wound responses. However, their contribution to downstream wound responses such as gene expression is not well understood. RESULTS: To identify genes regulated by WIPK and SIPK, the transcriptome of wounded WIPK/SIPK-suppressed plants was analyzed. Among the genes down-regulated in WIPK/SIPK-suppressed plants, the largest group consisted of those involved in the production of antimicrobial phytoalexins. Almost all genes involved in the biosynthesis of capsidiol, a major phytoalexin in tobacco, were transcriptionally induced by wounding in WIPK/SIPK-dependent and -independent manners. 5-epi-aristolochene synthase (EAS) is the committing enzyme for capsidiol synthesis, and the promoter of EAS4, a member of the EAS family, was analyzed. Reporter gene analysis revealed that at least two regions each 40-50 bp length were involved in activation of the EAS4 promoter by wounding, as well as by artificial activation of WIPK and SIPK. Unlike transcripts of the capsidiol synthesis genes, accumulation of EAS protein and capsidiol itself were not induced by wounding; however, wounding significantly enhanced their subsequent induction by a pathogen-derived elicitor. CONCLUSIONS: Our results suggest a so-called priming phenomenon since the induction of EAS by wounding is only visible at the transcript level. By inducing transcripts, not the proteins, of EAS and possibly other capsidiol synthesis genes at wound sites, plants can produce large quantities of capsidiol quickly if pathogens invade the wound site, whereas plants can minimize energy loss and avoid the cytotoxic effects of capsidiol where pathogens do not gain entry during wound healing.

Assessing the effects of quantitative host resistance on the life-history traits of sporulating parasites with growing lesions.

Assessing life-history traits of parasites on resistant hosts is crucial in evolutionary ecology. In the particular case of sporulating pathogens with growing lesions, phenotyping is difficult because one needs to disentangle properly pathogen spread from sporulation. By considering Phytophthora infestans on potato, we use mathematical modelling to tackle this issue and refine the assessment of pathogen response to quantitative host resistance. We elaborate a parsimonious leaf-scale model by convolving a lesion growth model and a sporulation function, after a latency period. This model is fitted to data obtained on two isolates inoculated on three cultivars with contrasted resistance level. Our results confirm a significant host-pathogen interaction on the various estimated traits, and a reduction of both pathogen spread and spore production, induced by host resistance. Most interestingly, we highlight that quantitative resistance also changes the sporulation function, the mode of which is significantly time-lagged. This alteration of the infectious period distribution on resistant hosts may have strong impacts on the dynamics of parasite populations, and should be considered when assessing the durability of disease control tactics based on plant resistance management. This inter-disciplinary work also supports the relevance of mechanistic models for analysing phenotypic data of plant-pathogen interactions.

Stacking three late blight resistance genes from wild species directly into African highland potato varieties confers complete field resistance to local blight races.

Considered responsible for one million deaths in Ireland and widespread famine in the European continent during the 1840s, late blight, caused by Phytophthora infestans, remains the most devastating disease of potato (Solanum tuberosum L.) with about 15%-30% annual yield loss in sub-Saharan Africa, affecting mainly smallholder farmers. We show here that the transfer of three resistance (R) genes from wild relatives [RB, Rpi-blb2 from Solanum bulbocastanum and Rpi-vnt1.1 from S. venturii] into potato provided complete resistance in the field over several seasons. We observed that the stacking of the three R genes produced a high frequency of transgenic events with resistance to late blight. In the field, 13 resistant transgenic events with the 3R-gene stack from the potato varieties 'Desiree' and 'Victoria' grew normally without showing pathogen damage and without any fungicide spray, whereas their non-transgenic equivalent varieties were rapidly killed. Characteristics of the local pathogen population suggest that the resistance to late blight may be long-lasting because it has low diversity, and essentially consists of the single lineage, 2_A1, which expresses the cognate avirulence effector genes. Yields of two transgenic events from 'Desiree' and 'Victoria' grown without fungicide to reflect small-scale farm holders were estimated to be 29 and 45 t/ha respectively. This represents a three to four-fold increase over the national average. Thus, these late blight resistant potato varieties, which are the farmers' preferred varieties, could be rapidly adopted and bring significant income to smallholder farmers in sub-Saharan Africa.

The Potato MAP3K StVIK Is Required for the Phytophthora infestans RXLR Effector Pi17316 to Promote Disease.

Plant pathogens deliver effectors to manipulate processes in their hosts, creating a suitable environment for invasion and proliferation. Yet, little is known about the host proteins that are targeted by effectors from filamentous pathogens. Here, we show that stable transgenic expression in potato (Solanum tuberosum) and transient expression in Nicotiana benthamiana of the arginine-any amino acid-leucine-arginine effector Pi17316 enhances leaf colonization by the late blight pathogen Phytophthora infestans Expression of Pi17316 also attenuates cell death triggered by the pathogen-associated molecular pattern Infestin1 (INF1), indicating that the effector suppresses pattern-triggered immunity. However, this effector does not attenuate cell death triggered by a range of resistance proteins, showing that it specifically suppresses INF1-triggered cell death (ICD). In yeast two-hybrid assays, Pi17316 interacts directly with the potato ortholog of VASCULAR HIGHWAY1-interacting kinase (StVIK), encoding a predicted MEK kinase (MAP3K). Interaction in planta was confirmed by coimmunoprecipitation and occurs at the plant plasma membrane. Virus-induced gene silencing of VIK in N. benthamiana attenuated P. infestans colonization, whereas transient overexpression of StVIK enhanced colonization, indicating that this host protein acts as a susceptibility factor. Moreover, VIK overexpression specifically attenuated ICD, indicating that it is a negative regulator of immunity. The abilities of Pi17316 to enhance P. infestans colonization or suppress ICD were compromised significantly in NbVIK-silenced plants, demonstrating that the effector activity of Pi17316 is mediated by this MAP3K. Thus, StVIK is exploited by P. infestans as a susceptibility factor to promote late blight disease.

MATE Transporter-Dependent Export of Hydroxycinnamic Acid Amides.

The ability of Arabidopsis thaliana to successfully prevent colonization by Phytophthora infestans, the causal agent of late blight disease of potato (Solanum tuberosum), depends on multilayered defense responses. To address the role of surface-localized secondary metabolites for entry control, droplets of a P. infestans zoospore suspension, incubated on Arabidopsis leaves, were subjected to untargeted metabolite profiling. The hydroxycinnamic acid amide coumaroylagmatine was among the metabolites secreted into the inoculum. In vitro assays revealed an inhibitory activity of coumaroylagmatine on P. infestans spore germination. Mutant analyses suggested a requirement of the p-coumaroyl-CoA:agmatine N4-p-coumaroyl transferase ACT for the biosynthesis and of the MATE transporter DTX18 for the extracellular accumulation of coumaroylagmatine. The host plant potato is not able to efficiently secrete coumaroylagmatine. This inability is overcome in transgenic potato plants expressing the two Arabidopsis genes ACT and DTX18. These plants secrete agmatine and putrescine conjugates to high levels, indicating that DTX18 is a hydroxycinnamic acid amide transporter with a distinct specificity. The export of hydroxycinnamic acid amides correlates with a decreased ability of P. infestans spores to germinate, suggesting a contribution of secreted antimicrobial compounds to pathogen defense at the leaf surface.

Genetic Structure and Subclonal Variation of Extant and Recent U.S. Lineages of Phytophthora infestans.

The oomycete Phytophthora infestans is an important plant pathogen on potato and tomato crops. We examined the genetic structure of extant 20th and 21st century U.S. lineages of P. infestans and compared them with populations from South America and Mexico to examine genetic relationships and potential sources of lineages. US-23, currently the most prevalent lineage detected in the United States, shared genetic similarity primarily with the BR-1 lineage identified in the 1990s from Bolivia and Brazil. Lineages US-8, US-14, and US-24, predominantly virulent on potato, formed a cluster distinct from other U.S. lineages. Many of the other U.S. lineages shared significant genetic similarity with Mexican populations. The US-1 lineage, dominant in the mid-20th century, clustered with US-1 lineages from Peru. A survey of the presence of RXLR effector PiAVR2 revealed that some lineages carried PiAVR2, its resistance-breaking variant PiAVR2-like, or both. Minimum spanning networks developed from simple sequence repeat genotype datasets from USABlight outbreaks clearly showed the expansion of US-23 over a 6-year time period and geographic substructuring of some lineages in the western United States. Many clonal lineages of P. infestans in the United States have come from introductions from Mexico, but the US-23 and US-1 lineages were most likely introduced from other sources.

Transgenic RXLR Effector PITG_15718.2 Suppresses Immunity and Reduces Vegetative Growth in Potato.

Phytophthora infestans causes the severe late blight disease of potato. During its infection process, P. infestans delivers hundreds of RXLR (Arg-x-Leu-Arg, x behalf of any one amino acid) effectors to manipulate processes in its hosts, creating a suitable environment for invasion and proliferation. Several effectors interact with host proteins to suppress host immunity and inhibit plant growth. However, little is known about how P. infestans regulates the host transcriptome. Here, we identified an RXLR effector, PITG_15718.2, which is upregulated and maintains a high expression level throughout the infection. Stable transgenic potato (Solanum tuberosum) lines expressing PITG_15718.2 show enhanced leaf colonization by P. infestans and reduced vegetative growth. We further investigated the transcriptional changes between three PITG_15718.2 transgenic lines and the wild type Désirée by using RNA sequencing (RNA-Seq). Compared with Désirée, 190 differentially expressed genes (DEGs) were identified, including 158 upregulated genes and 32 downregulated genes in PITG_15718.2 transgenic lines. Eight upregulated and nine downregulated DEGs were validated by real-time RT-PCR, which showed a high correlation with the expression level identified by RNA-Seq. These DEGs will help to explore the mechanism of PITG_15718.2-mediated immunity and growth inhibition in the future.

Comparative transcriptome analysis between resistant and susceptible tomato allows the identification of lncRNA16397 conferring resistance to Phytophthora infestans by co-expressing glutaredoxin.

The rapid development of omics sequencing technology has facilitated the identification of thousands of long non-coding (lnc)RNAs in plant species, but the role of lncRNAs in plant-pathogen interactions remains largely unexplored. We used comparative transcriptome analysis of Phytophthora infestans-resistant and -susceptible tomatoes to identify differentially expressed genes (DEGs) and lncRNAs (DELs), and examine lncRNA-mRNA networks. A total of 1037 DEGs and 688 DELs were identified between P. infestans-resistant and -susceptible tomatoes. The co-localization networks, including 128 DEGs and 127 DELs, were performed. We found that lncRNA16397 acted as an antisense transcript of SlGRX22 to regulate its expression, and also induced SlGRX21 expression when lncRNA16397 was overexpressed. In addition, disease symptoms and reactive oxygen species (ROS) accumulation in tomatoes overexpressing lncRNA16397 and SpGRX were fewer and lower than those in wild-type after P. infestans infection. This result suggests that tomato lncRNA16397 induces SlGRX expression to reduce ROS accumulation and alleviate cell membrane injury, resulting in enhanced resistance to P. infestans. Our results provide insight into lncRNAs involved in the response of tomato to P. infestans infection, demonstrate that the lncRNA16397-GRXs network is an important component of the P. infestans network in tomato, and provide candidates for breeding to enhance biotic stress-resistance in tomato.