Alternative splicing of a potato disease resistance gene maintains homeostasis between growth and immunity.

Plants possess a robust and sophisticated innate immune system against pathogens and must balance growth with rapid pathogen detection and defense. The intracellular receptors with nucleotide-binding leucine-rich repeat (NLR) motifs recognize pathogen-derived effector proteins and thereby trigger the immune response. The expression of genes encoding NLR receptors is precisely controlled in multifaceted ways. The alternative splicing (AS) of introns in response to infection is recurrently observed but poorly understood. Here we report that the potato (Solanum tuberosum) NLR gene RB undergoes AS of its intron, resulting in two transcriptional isoforms, which coordinately regulate plant immunity and growth homeostasis. During normal growth, RB predominantly exists as intron-retained isoform RB_IR, encoding a truncated protein containing only the N-terminus of the NLR. Upon late blight infection, the pathogen induces intron splicing of RB, increasing the abundance of RB_CDS, which encodes a full-length and active R protein. By deploying the RB splicing isoforms fused with a luciferase reporter system, we identified IPI-O1 (also known as Avrblb1), the RB cognate effector, as a facilitator of RB AS. IPI-O1 directly interacts with potato splicing factor StCWC15, resulting in altered localization of StCWC15 from the nucleoplasm to the nucleolus and nuclear speckles. Mutations in IPI-O1 that eliminate StCWC15 binding also disrupt StCWC15 re-localization and RB intron splicing. Thus, our study reveals that StCWC15 serves as a surveillance facilitator that senses the pathogen-secreted effector and regulates the trade-off between RB-mediated plant immunity and growth, expanding our understanding of molecular plant-microbe interactions.

Genotyping-by-Sequencing Reveals Population Differentiation and Linkage Disequilibrium in Alternaria linariae from Tomato.

Alternaria linariae is an economically important foliar pathogen that causes early blight disease in tomatoes. Understanding genetic diversity, population genetic structure, and evolutionary potential is crucial to contemplating effective disease management strategies. We leveraged genotyping-by-sequencing (GBS) technology to compare genome-wide variation in 124 isolates of Alternaria spp. (A. alternata, A. linariae, and A. solani) for comparative genome analysis and to test the hypotheses of genetic differentiation and linkage disequilibrium (LD) in A. linariae collected from tomatoes in western North Carolina. We performed a pangenome-aware variant calling and filtering with GBSapp and identified 53,238 variants conserved across the reference genomes of three Alternaria spp. The highest marker density was observed on chromosome 1 (7 Mb). Both discriminant analysis of principal components and Bayesian model-based STRUCTURE analysis of A. linariae isolates revealed three subpopulations with minimal admixture. The genetic differentiation coefficients (FST) within A. linariae subpopulations were similar and high (0.86), indicating that alleles in the subpopulations are fixed and the genetic structure is likely due to restricted recombination. Analysis of molecular variance indicated higher variation among populations (89%) than within the population (11%). We found long-range LD between pairs of loci in A. linariae, supporting the hypothesis of low recombination expected for a fungal pathogen with limited sexual reproduction. Our findings provide evidence of a high level of population genetic differentiation in A. linariae, which reinforces the importance of developing tomato varieties with broad-spectrum resistance to various isolates of A. linariae.

Spongospora subterranea f. sp. subterranea Affects Plant Susceptibility to Subsequent Pathogen Infections Under Controlled-Environment Conditions.

Spongospora subterranea f. sp. subterranea (Sss) is a soilborne potato pathogen responsible for causing powdery scab on tubers and galls on roots, reducing root water uptake through colonizing root hairs, and vectoring of Potato mop-top virus (PMTV). However, effects of Sss on overall plant susceptibilities against subsequent infections of potato pathogens above ground have not been previously reported. This study aimed to investigate the effects of Sss on root and tuber disease expression, yield, and susceptibilities to subsequent late blight and white mold infections across six potato varieties. Sss-infected Silverton plants had 28.3% less total tuber yield and 29% fewer tubers compared to noninfected Silverton plants. We did not find a correlation across the varieties between root colonization and root gall formation. Sss-infected Silverton plants were more susceptible to hemibiotrophic late blight and less susceptible to necrotrophic white mold. Sss infection also increased susceptibilities of Goldrush and Atlantic plants to white mold. We also evaluated prevalence of asymptomatic Sss infections across the six varieties. Between 50 and 92% of the asymptomatic tubers tested positive for Sss DNA, depending on the variety. Further research is required to understand the possibility and extent of these asymptomatic infections to the spread of Sss in the field. These findings highlight the complexity of Sss-host interactions and give precedence that the lack of disease expression does not necessarily indicate resistance of a variety to Sss.

Natural Genetic Diversity in the Potato Resistance Gene RB Confers Suppression Avoidance from Phytophthora Effector IPI-O4.

RB is a potato gene that provides resistance to a broad spectrum of genotypes of the late blight pathogen Phytophthora infestans. RB belongs to the CC-NB-LRR (coiled-coil, nucleotide-binding, leucine-rich repeat) class of resistance (R) genes, a major component of the plant immune system. The RB protein detects the presence of class I and II IPI-O effectors from P. infestans to initiate a hypersensitive resistance response, but this activity is suppressed in the presence of the Class III effector IPI-O4. Using natural genetic variation of RB within potato wild relatives, we identified two amino acids in the CC domain that alter interactions needed for suppression of resistance by IPI-O4. We have found that separate modification of these amino acids in RB can diminish or expand the resistance capability of this protein against P. infestans in both Nicotiana benthamiana and potato. Our results demonstrate that increased knowledge of the molecular mechanisms that determine resistance activation and R protein suppression by effectors can be utilized to tailor-engineer genes with the potential to provide increased durability.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Screening of Wild Potatoes Identifies New Sources of Late Blight Resistance.

Late blight (LB) of potato is considered one of the most devastating plant diseases in the world. Most cultivated potatoes are susceptible to this disease. However, wild relatives of potatoes are an excellent source of LB resistance. We screened 384 accessions of 72 different wild potato species available from the U.S. Potato GeneBank against the LB pathogen Phytophthora infestans in a detached leaf assay (DLA). P. infestans isolates US-23 and NL13316 were used in the DLA to screen the accessions. Although all plants in 273 accessions were susceptible, all screened plants in 39 accessions were resistant. Resistant and susceptible plants were found in 33 accessions. All tested plants showed a partial resistance phenotype in two accessions, segregation of resistant and partial resistant plants in nine accessions, segregation of partially resistant and susceptible plants in four accessions, and segregation of resistant, partially resistant, and susceptible individuals in 24 accessions. We found several species that were never before reported to be resistant to LB: Solanum albornozii, S. agrimoniifolium, S. chomatophilum, S. ehrenbergii, S. hypacrarthrum, S. iopetalum, S. palustre, S. piurae, S. morelliforme, S. neocardenasii, S. trifidum, and S. stipuloideum. These new species could provide novel sources of LB resistance. P. infestans clonal lineage-specific screening of selected species was conducted to identify the presence of RB resistance. We found LB resistant accessions in Solanum verrucosum, Solanum stoloniferum, and S. morelliforme that were susceptible to the RB overcoming isolate NL13316, indicating the presence of RB-like resistance in these species.

Overexpression of a modified eIF4E regulates potato virus Y resistance at the transcriptional level in potato.

BACKGROUND: Potato virus Y (PVY) is a major pathogen of potatoes with major impact on global agricultural production. Resistance to PVY can be achieved by engineering potatoes to express a recessive, resistant allele of eukaryotic translation initiation factor eIF4E, a host dependency factor essential to PVY replication. Here we analyzed transcriptome changes in eIF4E over-expressing potatoes to shed light on the mechanism underpinning eIF4E-mediated recessive PVY resistance. RESULTS: As anticipated, modified eIF4E-expressing potatoes demonstrated a high level of resistance, eIF4E expression, and an unexpected suppression of the susceptible allele transcript, likely explaining the bulk of the potent antiviral phenotype. In resistant plants, we also detected marked upregulation of genes involved in cell stress responses. CONCLUSIONS: Our results reveal a previously unanticipated second layer of signaling attributable to eIF4E regulatory control, and potentially relevant to establishment of a broader, more systematic antiviral host defense.

Gene Genealogies Reveal High Nucleotide Diversity and Admixture Haplotypes Within Three Alternaria Species Associated with Tomato and Potato.

Early blight (EB) and leaf blight are two destructive diseases of tomato in North Carolina (NC), caused by Alternaria linariae and A. alternata, respectively. During the last decade, EB caused by A. solani has increased in potato-producing areas in Wisconsin (WI). We collected 152 isolates of three Alternaria spp. associated with tomato and potato in NC and WI and used the gene genealogical approach to compare the genetic relationships among them. Two nuclear genes: the glyceraldehyde-3-phosphate dehydrogenase (GPDH), RNA polymerase second largest subunit (RPB2), and the rDNA internal transcribed spacer (ITS) region of these isolates were sequenced. Besides, sequences of the GPDH locus from international isolates described in previous studies were included for comparison purposes. A set of single nucleotide polymorphisms was assembled to identify locus-specific and species-specific haplotypes. Nucleotide diversity varied among gene sequences and species analyzed. For example, the estimates of nucleotide diversity and Watterson's theta were higher in A. alternata than in A. linariae and A. solani. There was little or no polymorphisms in the ITS sequences and thus restricted haplotype placement. The RPB2 sequences were less informative to detect haplotype diversity in A. linariae and A. solani, yet six haplotypes were detected in A. alternata. The GPDH sequences enabled strongly supported phylogenetic inferences with the highest haplotype diversity and belonged to five haplotypes (AaH1 to AaH5), which consisted of only A. alternata from NC. However, 13 haplotypes were identified within and among A. linariae and A. solani sequences. Among them, six (AsAlH1 to AsAlH6) were identical to previously reported haplotypes in global samples and the remaining were new haplotypes. The most divergent haplotypes were AaH1, AsAlH2/AsAlH3, and AsAlH4 and consisted exclusively of A. alternata, A. linariae, and A. solani, respectively. Neutrality tests suggested an excess of mutations and population expansion, and selection may play an important role in nucleotide diversity of Alternaria spp.

HCPro Suppression of Callose Deposition Contributes to Strain-Specific Resistance Against Potato Virus Y.

Potato virus Y (PVY; Potyviridae) is a continuing challenge for potato production owing to the increasing popularity of strain-specific resistant cultivars. Hypersensitive resistance (HR) is one type of plant defense responses to restrict virus spread. In many potato cultivars, such as cultivar Premier Russet (PR), local necrosis at the site of infection protects against the most common PVYO strain, but the HR often fails to restrain necrotic strains, which spread systemically. Here, we established the role of callose accumulation in the strain-specific resistance responses to PVY infection. We first uncovered that PVY, independent of the strain, is naturally capable of suppressing pathogenesis-related callose formation in a susceptible host. Such activity can be dissociated from viral replication by the transient expression of the viral-encoded helper component proteinase (HCPro) protein, identifying it as the pathogen elicitor. However, unlike the necrotic strain, PVYO and its corresponding HCPro are unable to block callose accumulation in resistant PR potatoes, in which we observed an abundance of callose deposition and the inability of the virus to spread. The substitution of eight amino acid residues within the HCPro C-terminal region that differ between PVYO and PVYN strains and were previously shown to be responsible for eliciting the HR response, are sufficient to restore the ability of HCProO to suppress callose accumulation, despite the resistant host background, in line with a new viral function in pathogenicity.

Distribution and Stability of Quinone Outside Inhibitor Fungicide Resistance in Populations of Potato Pathogenic Alternaria spp. in Wisconsin.

Quinone outside inhibitor (QoI) fungicides have been an important class in managing potato early blight caused by Alternaria solani and brown spot caused by A. alternata. Because of the single-site mode of action character of QoI fungicides, which are relied on for management of diseases in Wisconsin, and the abundant asexual conidia production of the Alternaria species, pathogen isolates with QoI resistance have been detected after just a few years of QoI fungicide usage in commercial production fields. Resistance to QoIs has been attributed to amino acid substitutions F129L and G143A in cytochrome b of A. solani and A. alternata, respectively, as a result of point mutations. The aim of this study was to assess Alternaria populations in Wisconsin for QoI resistance before and after fungicide applications in order to evaluate resistance stability. A TaqMan single nucleotide polymorphism genotyping assay was designed based on the sequences of the cytochrome b gene from Alternaria isolates collected in Wisconsin to profile QoI resistance in Alternaria populations as well as to explore factors that may influence frequency of QoI resistance in the pathogen populations. This assay successfully identified the mutations conferring QoI resistance in isolates collected from four locations each year from 2015 to 2017. During the course of this study, the frequency of A. solani isolates with the F129L mutation was consistently high and showed primarily the TTA mutation type. The frequency of A. alternata isolates with the G143A mutation started relatively low and increased at the end of the production season in each year (P = 0.0109, P = 0.2083, and P = 0.0159). A potato field managed without use of QoI fungicides showed a significantly lower (P < 0.05) frequency of A. alternata isolates carrying G143A than conventionally managed potato fields. The overall frequency of A. alternata isolates carrying G143A in the four locations was similar over the 3 years (P = 0.2971). The QoI resistance characteristics of the isolates were stable even when QoI selection pressure was removed for at least five subculture transfers, and the mutation types of codons 129 and 143 in the cytochrome b gene in A. solani and A. alternata, respectively, remained the same. This indicated that the application of QoIs in the field is not the sole factor responsible for the variation of the frequency of QoI resistance in the pathogen populations.

Phytophthora infestans Effectors IPI-O1 and IPI-O4 Each Contribute to Pathogen Virulence.

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, is one of the most destructive plant diseases. Despite decades of intensive breeding efforts, it remains a threat to potato production worldwide, because newly evolved pathogen strains have overcome major resistance genes quickly. The RB protein, from the diploid wild potato species Solanum bulbocastanum, confers partial resistance to most P. infestans strains through its recognition of members of the corresponding pathogen effector protein family IPI-O. IPI-O comprises a multigene family and while some variants are recognized by RB to elicit host resistance (e.g., IPI-O1 and IPI-O2), others are able to elude detection (e.g., IPI-O4). IPI-O1 is almost ubiquitous in global P. infestans strains while IPI-O4 is more rare. No direct experimental evidence has been shown to demonstrate the effect of IPI-O on pathogen virulence in the P. infestans-potato pathosystem. Here, our work has demonstrated that in planta expression of both IPI-O1 and IPI-O4 increases P. infestans aggressiveness resulting in enlarged lesions in potato leaflets. We have previously shown that IPI-O4 has gained the ability to suppress the hypersensitive response induced by IPI-O1 in the presence of RB. In this study, our work has shown that this gain-of-function of IPI-O4 does not compromise its virulence effect, as IPI-O4 overexpression results in larger lesions than IPI-O1. We have also found that higher expression of IPI-O effectors correlates with enlarged lesions, indicating that IPI-O can contribute to virulence quantitatively. In summary, this study has provided accurate and valuable information on IPI-O's virulence effect on the potato host.

The IRE1/bZIP60 Pathway and Bax Inhibitor 1 Suppress Systemic Accumulation of Potyviruses and Potexviruses in Arabidopsis and Nicotiana benthamiana Plants.

The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor. When activated, it splices the bZIP60 mRNA, producing a truncated transcription factor that upregulates genes involved in the unfolded protein response. Bax inhibitor 1 (BI-1) is another ER stress sensor that regulates cell death in response to environmental assaults. The potyvirus 6K2 and potexvirus TGB3 proteins are known to reside in the ER, serving, respectively, as anchors for the viral replicase and movement protein complex. This study used green fluorescent protein (GFP)-tagged Turnip mosaic virus (TuMV), Plantago asiatica mosaic virus (PlAMV), Potato virus Y (PVY), and Potato virus X (PVX) to determine that the IRE1/bZIP60 pathway and BI-1 machinery are induced early in virus infection in Arabidopsis thaliana, Nicotiana benthamiana, and Solanum tuberosum. Agrodelivery of only the potyvirus 6K2 or TGB3 genes into plant cells activated bZIP60 and BI-1 expression in Arabidopsis thaliana, N. benthamiana, and S. tuberosum. Homozygous ire1a-2, ire1b-4, and ire1a-2/ire1b-4 mutant Arabidopsis plants were inoculated with TuMV-GFP or PlAMV-GFP. PlAMV accumulates to a higher level in ire1a-2 or ire1a-2/ire1b-4 mutant plants than in ire1b-4 or wild-type plants. TuMV-GFP accumulates to a higher level in ire1a-2, ire1b-4, or ire1a-2/ire1b-4 compared with wild-type plants, suggesting that both isoforms contribute to TuMV-GFP infection. Gene silencing was used to knock down bZIP60 and BI-1 expression in N. benthamiana. PVX-GFP and PVY-GFP accumulation was significantly elevated in these silenced plants compared with control plants. This study demonstrates that two ER stress pathways, namely IRE1/bZIP60 and the BI-1 pathway, limit systemic accumulation of potyvirus and potexvirus infection. Silencing BI-1 expression also resulted in systemic necrosis. These data suggest that ER stress-activated pathways, led by IRE1 and BI-1, respond to invading potyvirus and potexviruses to restrict virus infection and enable physiological changes enabling plants to tolerate virus assault.

Molecular determinants of resistance activation and suppression by Phytophthora infestans effector IPI-O.

Despite intensive breeding efforts, potato late blight, caused by the oomycete pathogen Phytophthora infestans, remains a threat to potato production worldwide because newly evolved pathogen strains have consistently overcome major resistance genes. The potato RB gene, derived from the wild species Solanum bulbocastanum, confers resistance to most P. infestans strains through recognition of members of the pathogen effector family IPI-O. While the majority of IPI-O proteins are recognized by RB to elicit resistance (e.g. IPI-O1, IPI-O2), some family members are able to elude detection (e.g. IPI-O4). In addition, IPI-O4 blocks recognition of IPI-O1, leading to inactivation of RB-mediated programmed cell death. Here, we report results that elucidate molecular mechanisms governing resistance elicitation or suppression of RB by IPI-O. Our data indicate self-association of the RB coiled coil (CC) domain as well as a physical interaction between this domain and the effectors IPI-O4 and IPI-O1. We identified four amino acids within IPI-O that are critical for interaction with the RB CC domain and one of these amino acids, at position 129, determines hypersensitive response (HR) elicitation in planta. IPI-O1 mutant L129P fails to induce HR in presence of RB while IPI-O4 P129L gains the ability to induce an HR. Like IPI-O4, IPI-O1 L129P is also able to suppress the HR mediated by RB, indicating a critical step in the evolution of this gene family. Our results point to a model in which IPI-O effectors can affect RB function through interaction with the RB CC domain.

Multifunctionality of AsCFEM6 and AsCFEM12 effectors from the potato early blight pathogen Alternaria solani.

Plant pathogens secrete fungal-specific common in several fungal extracellular membrane (CFEM) effectors to manipulate host immunity and contribute to their virulence. Little is known about effectors and their functions in Alternaria solani, the necrotrophic fungal pathogen causing potato early blight. To identify candidate CFEM effector genes, we mined A. solani genome databases. This led to the identification of 12 genes encoding CFEM proteins (termed AsCFEM1-AsCFEM12) and 6 of them were confirmed to be putative secreted effectors. In planta expression revealed that AsCFEM6 and AsCFEM12 have elicitor function that triggers plant defense response including cell death in different botanical families. Targeted gene disruption of AsCFEM6 and AsCFEM12 resulted in a change in spore development, significant reduction of virulence on potato and eggplant susceptible cultivars, increased resistance to fungicide stress, variation in iron acquisition and utilization, and the involvement in 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway. Using maximum likelihood method, we found that positive selection likely caused the polymorphism within AsCFEM6 and AsCFEM12 homologs in different Alternaria spp. Site-directed mutagenesis analysis indicated that positive selection sites within their CFEM domains are required for cell death induction in Nicotiana benthamiana and are critical for response to abiotic stress in yeast. These results demonstrate that AsCFEM effectors possess additional functions beyond their roles in host plant immune response and pathogen virulence.

A survey of the Sli gene in wild and cultivated potato.

Inbred-hybrid breeding of diploid potatoes necessitates breeding lines that are self-compatible. One way of incorporating self-compatibility into incompatible cultivated potato (Solanum tuberosum) germplasm is to introduce the S-locus inhibitor gene (Sli), which functions as a dominant inhibitor of gametophytic self-incompatibility. To learn more about Sli diversity and function in wild species relatives of cultivated potato, we obtained Sli gene sequences that extended from the 5'UTR to the 3'UTR from 133 individuals from 22 wild species relatives of potato and eight diverse cultivated potato clones. DNA sequence alignment and phylogenetic trees based on genomic and protein sequences show that there are two highly conserved groups of Sli sequences. DNA sequences in one group contain the 533 bp insertion upstream of the start codon identified previously in self-compatible potato. The second group lacks the insertion. Three diploid and four polyploid individuals of wild species collected from geographically disjointed localities contained Sli with the 533 bp insertion. For most of the wild species clones examined, however, Sli did not have the insertion. Phylogenetic analysis indicated that Sli sequences with the insertion, in wild species and in cultivated clones, trace back to a single origin. Some diploid wild potatoes that have Sli with the insertion were self-incompatible and some wild potatoes that lack the insertion were self-compatible. Although there is evidence of positive selection for some codon positions in Sli, there is no evidence of diversifying selection at the gene level. In silico analysis of Sli protein structure did not support the hypothesis that amino acid changes from wild-type (no insertion) to insertion-type account for changes in protein function. Our study demonstrated that genetic factors besides the Sli gene must be important for conditioning a switch in the mating system from self-incompatible to self-compatible in wild potatoes.

Decreased defense gene expression in tolerance versus resistance to Verticillium dahliae in potato.

Verticillium dahliae Kleb., a soil-borne fungus that colonizes vascular tissues, induces wilting, chlorosis and early senescence in potato. Difference in senescence timing found in two diploid potato clones, 07506-01 and 12120-03, was studied and genetic variation in response to V. dahliae infection was identified as a causal factor. The clone, 07506-01, was infected with V. dahliae but did not develop symptoms, indicating tolerance to the pathogen. The other diploid clone, 12120-03 had low levels of pathogen with infection and moderate symptoms indicating partial resistance. 07506-01 was found to carry two susceptible alleles of the Ve2 gene and 12120-03 carried one Ve2 resistant and one susceptible allele. Infected leaves of the two clones were compared using gene expression profiling with the Potato Oligonucleotide Chip Initiative (POCI) microrarray. The results provide further evidence for differences in response of the two clones to infection with V. dahliae. Chlorophyll biosynthesis was higher in the tolerant 07506-01 compared to partially resistant 12120-03. On the other hand, expression of fungal defense genes, Ve resistance genes and defense phytohormone biosynthetic enzyme genes was decreased in 07506-01 compared to 12120-03 suggesting defense responses were suppressed in tolerance compared to resistance. Transcription factor gene expression differences pointed to the WRKY family as potential regulators of V. dahliae responses in potato.

Phenotypic characterization of potato late blight resistance mediated by the broad-spectrum resistance gene RB.

The potato gene RB, cloned from the wild potato species Solanum bulbocastanum, confers partial resistance to late blight, caused by the oomycete pathogen Phytophthora infestans. In order to better characterize this partial resistance phenotype, we have compared host resistance responses mediated by RB with those mediated by the S. demissum-derived R gene R9, which confers immunity to P. infestans carrying the corresponding avirulence gene avrR9. We found that both RB and R9 genes were capable of eliciting a hypersensitive cell death response (HR). However, in RB plants, the pathogen escaped HR lesions and continued to grow beyond the inoculation sites. We also found that callose deposition was negatively correlated with resistance levels in tested plants. Transcription patterns of pathogenesis-related (PR) genes PR-1 basic, PR-2 acidic, and PR-5 indicated that P. infestans inoculation induced transcription of these defense-related genes regardless of the host genotype; however, transcription was reduced in both the susceptible and partially resistant plants later in the infection process but remained elevated in the immune host. Most interestingly, transcription of the HR-associated gene Hin1 was suppressed in both Katahdin and RB-transgenic Katahdin but not in R9 4 days after inoculation. Together, this suggests that suppression of certain defense-related genes may allow P. infestans to spread beyond the site of infection in the partially resistant host despite elicitation of hypersensitive cell death.

Phytophthora infestans (Late blight) Infection Assay in a Detached Leaf of Potato.

Phytophthora infestans is a hemibiotroph oomycete that primarily infects potato and tomato. It infects stems, leaves, and tubers and fruits of potato and tomato. High throughput and reproducible infection assays are prerequisites to find sources of resistance in any crop. In this protocol, we describe a detached leaf assay (DLA) for conducting the virulence assay of P. infestans in potato leaves. A late blight infection assay using a potato detached leaf is a semi-high throughput assay in which hundreds of plants can be screened in a laboratory setting.

Genetic diversity and population structure of Alternaria species from tomato and potato in North Carolina and Wisconsin.

Early blight (EB) caused by Alternaria linariae or Alternaria solani and leaf blight (LB) caused by A. alternata are economically important diseases of tomato and potato. Little is known about the genetic diversity and population structure of these pathogens in the United States. A total of 214 isolates of A. alternata (n = 61), A. linariae (n = 96), and A. solani (n = 57) were collected from tomato and potato in North Carolina and Wisconsin and grouped into populations based on geographic locations and tomato varieties. We exploited 220 single nucleotide polymorphisms derived from DNA sequences of 10 microsatellite loci to analyse the population genetic structure between species and between populations within species and infer the mode of reproduction. High genetic variation and genotypic diversity were observed in all the populations analysed. The null hypothesis of the clonality test based on the index of association [Formula: see text] was rejected, and equal frequencies of mating types under random mating were detected in some studied populations of Alternaria spp., suggesting that recombination can play an important role in the evolution of these pathogens. Most genetic differences were found between species, and the results showed three distinct genetic clusters corresponding to the three Alternaria spp. We found no evidence for clustering of geographic location populations or tomato variety populations. Analyses of molecular variance revealed high (> 85%) genetic variation within individuals in a population, confirming a lack of population subdivision within species. Alternaria linariae populations harboured more multilocus genotypes (MLGs) than A. alternata and A. solani populations and shared the same MLG between populations within a species, which was suggestive of gene flow and population expansion. Although both A. linariae and A. solani can cause EB on tomatoes and potatoes, these two species are genetically differentiated. Our results provide new insights into the evolution and structure of Alternaria spp. and can lead to new directions in optimizing management strategies to mitigate the impact of these pathogens on tomato and potato production in North Carolina and Wisconsin.

Genome-wide identification and expression profiling of basic leucine zipper transcription factors following abiotic stresses in potato (Solanum tuberosum L.).

Potato (Solanum tuberosum L.) is an important food crop that is grown and consumed worldwide. The growth and productivity of this crop are severely affected by various abiotic stresses. Basic leucine zipper (bZIP) transcription factors (TFs) in plants are well known for their function during growth and development. However, systematic and in-depth identification and functional characterization of the bZIP gene family of potato is lacking. In the current study, we identified a total of 90 bZIPs (StbZIP) distributed on 12 linkage groups of potato. Based on the previous functional annotation and classification of bZIPs in Arabidopsis, wheat, and rice, a phylogenetic tree of potato bZIPs was constructed and genes were categorized into various functional groups (A to I, S, and U) as previously annotated in Arabidopsis thaliana. Analyses of the transcript sequence (RNA-seq) data led to identifying a total of 18 candidate StbZIPs [four in roots, eight in the tuber, six in mesocarp and endocarp] that were expressed in a tissue-specific manner. Differential expression analysis under the various abiotic conditions (salt, mannitol, water, and heat stress) and treatment with phytohormones (ABA, GA, IAA, and BAP) led to the identification of forty-two [thirteen under salt stress, two under mannitol stress, ten under water stress, and eighteen under heat stress], and eleven [eight and three StbZIPs upon treatment with ABA, and IAA, respectively] candidate StbZIPs, respectively. Using sequence information of candidate StbZIPs, a total of 22 SSR markers were also identified in this study. In conclusion, the genome-wide identification analysis coupled with RNA-Seq expression data led to identifying candidate StbZIPs, which are dysregulated, and may play a pivotal role under various abiotic stress conditions. This study will pave the way for future functional studies using forward and reverse genetics to improve abiotic stress tolerance in potato.

Biochemical evaluation of resistance responses of potato to different isolates of Alternaria solani.

The resistance phenotypes of nine potato cultivars to five isolates of Alternaria solani, causal agent of early blight, were studied after inoculation and growth under greenhouse conditions. We identified potato cultivars with both susceptible and resistant phenotypes as well as A. solani isolates with varying degrees of aggressiveness. Two potato cultivars and two pathogen isolates were selected for biochemical analysis of phenol production and peroxidase activity after inoculation. Phenol compounds were evaluated 2, 4, 6, and 8 days after inoculation, while peroxidase activities were monitored daily for 10 days. Native polyacrylamide electrophoresis was used to identify one protein with peroxidase activity in extracts taken 6 days after inoculation. Significantly higher peroxidase activity as well as total phenol content in potato was correlated with resistance in the Iranian potato cultivar Diamond. Variability of responses within the same cultivar to different isolates of A. solani suggests genotypic diversity between isolates that results in phenotypic diversity for pathogen aggressiveness.