Transcriptome Analysis of a Cotton Cultivar Provides Insights into the Differentially Expressed Genes Underlying Heightened Resistance to the Devastating Verticillium Wilt.

Cotton is an important economic crop worldwide. Verticillium wilt (VW) caused by Verticillium dahliae (V. dahliae) is a serious disease in cotton, resulting in massive yield losses and decline of fiber quality. Breeding resistant cotton cultivars is an efficient but elaborate method to improve the resistance of cotton against V. dahliae infection. However, the functional mechanism of several excellent VW resistant cotton cultivars is poorly understood at present. In our current study, we carried out RNA-seq to discover the differentially expressed genes (DEGs) in the roots of susceptible cotton Gossypium hirsutum cultivar Junmian 1 (J1) and resistant cotton G.hirsutum cultivar Liaomian 38 (L38) upon Vd991 inoculation at two time points compared with the mock inoculated control plants. The potential function of DEGs uniquely expressed in J1 and L38 was also analyzed by GO enrichment and KEGG pathway associations. Most DEGs were assigned to resistance-related functions. In addition, resistance gene analogues (RGAs) were identified and analyzed for their role in the heightened resistance of the L38 cultivar against the devastating Vd991. In summary, we analyzed the regulatory network of genes in the resistant cotton cultivar L38 during V. dahliae infection, providing a novel and comprehensive insight into VW resistance in cotton.

Cotton CC-NBS-LRR Gene GbCNL130 Confers Resistance to Verticillium Wilt Across Different Species.

Verticillium wilt (VW) is a destructive disease in cotton caused by Verticillium dahliae and has a significant impact on yield and quality. In the absence of safe and effective chemical control, VW is difficult to manage. Thus, at present, developing resistant varieties is the most economical and effective method of controlling Verticillium wilt of cotton. The CC-NBS-LRR (CNL) gene family is an important class of plant genes involved in disease resistance. This study identified 141 GbCNLs in Gossypium barbadense genome, with 37.5% (53 genes) GbCNLs enriched in 12 gene clusters (GC01-GC12) based on gene distribution in the chromosomes. Especially, seven GbCNLs from two largest clusters (GC11 and GC12) were significantly upregulated in the resistant cultivar (Hai No. 7124) and the susceptible (Giza No. 57). Virus-induced gene silencing of GbCNL130 in G. barbadense, one typical gene in the gene cluster 12 (GC12), significantly altered the response to VW, compromising plant resistance to V. dahliae. In contrast, GbCNL130 overexpression significantly increased the resistance to VW in the wild-type Arabidopsis thaliana. Based on our research findings presented here, we conclude that GbCNL130 promotes resistance to VW by activating the salicylic acid (SA)-dependent defense response pathway resulting in strong accumulation of reactive oxygen species and upregulation of pathogenesis-related (PR) genes. In conclusion, our study resulted in the discovery of a new CNL resistance gene in cotton, GbCNL130, that confers resistance to VW across different hosts.

Lysin Motif (LysM) Proteins: Interlinking Manipulation of Plant Immunity and Fungi.

The proteins with lysin motif (LysM) are carbohydrate-binding protein modules that play a critical role in the host-pathogen interactions. The plant LysM proteins mostly function as pattern recognition receptors (PRRs) that sense chitin to induce the plant's immunity. In contrast, fungal LysM blocks chitin sensing or signaling to inhibit chitin-induced host immunity. In this review, we provide historical perspectives on plant and fungal LysMs to demonstrate how these proteins are involved in the regulation of plant's immune response by microbes. Plants employ LysM proteins to recognize fungal chitins that are then degraded by plant chitinases to induce immunity. In contrast, fungal pathogens recruit LysM proteins to protect their cell wall from hydrolysis by plant chitinase to prevent activation of chitin-induced immunity. Uncovering this coevolutionary arms race in which LysM plays a pivotal role in manipulating facilitates a greater understanding of the mechanisms governing plant-fungus interactions.

Hormone Signaling and Its Interplay With Development and Defense Responses in Verticillium-Plant Interactions.

Soilborne plant pathogenic species in the fungal genus Verticillium cause destructive Verticillium wilt disease on economically important crops worldwide. Since R gene-mediated resistance is only effective against race 1 of V. dahliae, fortification of plant basal resistance along with cultural practices are essential to combat Verticillium wilts. Plant hormones involved in cell signaling impact defense responses and development, an understanding of which may provide useful solutions incorporating aspects of basal defense. In this review, we examine the current knowledge of the interplay between plant hormones, salicylic acid, jasmonic acid, ethylene, brassinosteroids, cytokinin, gibberellic acid, auxin, and nitric oxide, and the defense responses and signaling pathways that contribute to resistance and susceptibility in Verticillium-host interactions. Though we make connections where possible to non-model systems, the emphasis is placed on Arabidopsis-V. dahliae and V. longisporum interactions since much of the research on this interplay is focused on these systems. An understanding of hormone signaling in Verticillium-host interactions will help to determine the molecular basis of Verticillium wilt progression in the host and potentially provide insight on alternative approaches for disease management.

Overexpression of Arabidopsis microRNA167 induces salicylic acid-dependent defense against Pseudomonas syringae through the regulation of its targets ARF6 and ARF8.

microRNAs are powerful regulators of growth, development, and stress responses in plants. The Arabidopsis thaliana microRNA miR167 was previously found to regulate diverse processes including flower development, root development, and response to osmotic stress by controlling the patterns of expression of its target genes AUXIN RESPONSE FACTOR 6 (ARF6), ARF8, and IAA-Ala RESISTANT 3. Here, we report that miR167 also modulates defense against pathogens through ARF6 and ARF8. miR167 is differentially expressed in response to the bacterial pathogen Pseudomonas syringae, and overexpression of miR167 confers very high levels of resistance. This resistance appears to be due to suppression of auxin responses and is partially dependent upon salicylic acid signaling, and also depends upon altered stomatal behavior in these plants. Closure of stomata upon the detection of P. syringae is an important aspect of the basal defense response, as it prevents bacterial cells from entering the leaf interior and causing infection. Plants overexpressing miR167 constitutively maintain small stomatal apertures, resulting in very high resistance when the pathogen is inoculated onto the leaf surface. Additionally, the systemic acquired resistance (SAR) response is severely compromised in plants overexpressing miR167, in agreement with previous work showing that the activation of SAR requires intact auxin signaling responses. This work highlights a new role for miR167, and also emphasizes the importance of hormonal balance in short- and long-term defense and of stomata as an initial barrier to pathogen entry.

An Arabidopsis DISEASE RELATED NONSPECIFIC LIPID TRANSFER PROTEIN 1 is required for resistance against various phytopathogens and tolerance to salt stress.

Synchronous and timely regulation of multiple genes results in an effective defense response that decides the fate of the host when challenged with pathogens or unexpected changes in environmental conditions. One such gene, which is downregulated in response to multiple bacterial pathogens, is a putative nonspecific lipid transfer protein (nsLTP) of unknown function that we have named DISEASE RELATED NONSPECIFIC LIPID TRANSFER PROTEIN 1 (DRN1). We show that upon pathogen challenge, DRN1 is strongly downregulated, while a putative DRN1-targeting novel microRNA (miRNA) named DRN1 Regulating miRNA (DmiR) is reciprocally upregulated. Furthermore, we provide evidence that DRN1 is required for defense against bacterial and fungal pathogens as well as for normal seedling growth under salinity stress. Although nsLTP family members from different plant species are known to be a significant source of food allergens and are often associated with antimicrobial properties, our knowledge on the biological functions and regulation of this gene family is limited. Our current work not only sheds light on the mechanism of regulation but also helps in the functional characterization of DRN1, a putative nsLTP family member of hitherto unknown function.

The Arabidopsis SENESCENCE-ASSOCIATED GENE 13 Regulates Dark-Induced Senescence and Plays Contrasting Roles in Defense Against Bacterial and Fungal Pathogens.

SENESCENCE-ASSOCIATED GENE 13 (SAG13) of Arabidopsis is a widely conserved gene of unknown function that has been extensively used as a marker of plant senescence. SAG13 induction occurs during plant cell death processes, including senescence and hypersensitive response, a type of programmed cell death that occurs in response to pathogens. This implies that SAG13 expression is regulated through at least two different signaling pathways affecting these two different processes. Our work highlights a contrasting role for SAG13 in regulating resistance against disease-causing biotrophic bacterial and necrotrophic fungal pathogens with contrasting infection strategies. We provide further evidence that SAG13 is not only induced during oxidative stress but also plays a role in protecting the plant against other stresses. SAG13 is also required for normal seed germination, seedling growth, and anthocyanin accumulation. The work presented here provides evidence for the role of SAG13 in regulating multiple plant processes including senescence, defense, seed germination, and abiotic stress responses. SAG13 is a valuable molecular marker for these processes and is conserved in multiple plant species, and this knowledge has important implications for crop improvement.

Measurements of Aerial Spore Load by qPCR Facilitates Lettuce Downy Mildew Risk Advisement.

The lettuce downy mildew pathogen, Bremia lactucae, is an obligate oomycete that causes extensive produce losses. Initial chlorotic symptoms that severely reduce the market value of the produce are followed by the appearance of white, downy sporulation on the abaxial side of the leaves. These spores become airborne and disseminate the pathogen. Controlling lettuce downy mildew has relied on repeated fungicide applications to prevent outbreaks. However, in addition to direct economic costs, heterogeneity and rapid adaptation of this pathogen to repeatedly applied fungicides has led to the development of fungicide-insensitivity in the pathogen. We deployed a quantitative PCR assay-based detection method using a species-specific DNA target for B. lactucae coupled with a spore trap system to measure airborne B. lactucae spore loads within three commercial fields that each contained experimental plots, designated EXP1 to EXP3. Based upon these measurements, when the spore load in the air reached a critical level (8.548 sporangia per m3 air), we advised whether or not to apply fungicides on a weekly basis within EXP1 to EXP3. This approach saved three sprays in EXP1, and one spray each in EXP2 and EXP3 without a significant increase in disease incidence. The reduction in fungicide applications to manage downy mildew can decrease lettuce production costs while slowing the development of fungicide resistance in B. lactucae by eliminating unnecessary fungicide applications.

Arabidopsis defense mutant ndr1-1 displays accelerated development and early flowering mediated by the hormone gibberellic acid.

NONRACE-SPECIFIC DISEASE RESISTANCE (NDR1) is a widely characterized gene that plays a key role in defense against multiple bacterial, fungal, oomycete and nematode plant pathogens. NDR1 is required for activation of resistance by multiple NB and LRR-containing (NLR) protein immune sensors and contributes to basal defense. The role of NDR1 in positively regulating salicylic acid (SA)-mediated plant defense responses is well documented. However, ndr1-1 plants flower earlier and show accelerated development in comparison to wild type (WT) Arabidopsis plants, indicating that NDR1 is a negative regulator of flowering and growth. Exogenous application of gibberellic acid (GA) further accelerates the early flowering phenotype in ndr1-1 plants, while the GA biosynthesis inhibitor paclobutrazol attenuated the early flowering phenotype of ndr1-1, but not to WT levels, suggesting partial resistance to paclobutrazol and enhanced GA response in ndr1-1 plants. Mass spectroscopy analyses confirmed that ndr1-1 plants have 30-40% higher levels of GA3 and GA4, while expression of various GA metabolic genes and major flowering regulatory genes is also altered in the ndr1-1 mutant. Taken together this study provides evidence of crosstalk between the ndr1-1-mediated defense and GA-regulated developmental programs in plants.

Genome-Wide Identification and Functional Analyses of the CRK Gene Family in Cotton Reveals GbCRK18 Confers Verticillium Wilt Resistance in Gossypium barbadense.

Cysteine-rich receptor-like kinases (CRKs) are a large subfamily of plant receptor-like kinases that play a critical role in disease resistance in plants. However, knowledge about the CRK gene family in cotton and its function against Verticillium wilt (VW), a destructive disease caused by Verticillium dahliae that significantly reduces cotton yields is lacking. In this study, we identified a total of 30 typical CRKs in a Gossypium barbadense genome (GbCRKs). Eleven of these (>30%) are located on the A06 and D06 chromosomes, and 18 consisted of 9 paralogous pairs encoded in the A and D subgenomes. Phylogenetic analysis showed that the GbCRKs could be classified into four broad groups, the expansion of which has probably been driven by tandem duplication. Gene expression profiling of the GbCRKs in resistant and susceptible cotton cultivars revealed that a phylogenetic cluster of nine of the GbCRK genes were up-regulated in response to V. dahliae infection. Virus-induced gene silencing of each of these nine GbCRKs independently revealed that the silencing of GbCRK18 was sufficient to compromise VW resistance in G. barbadense. GbCRK18 expression could be induced by V. dahliae infection or jasmonic acid, and displayed plasma membrane localization. Therefore, our expression analyses indicated that the CRK gene family is differentially regulated in response to Verticillium infection, while gene silencing experiments revealed that GbCRK18 in particular confers VW resistance in G. barbadense.

Detection of Latent Peronospora effusa Infections in Spinach.

Downy mildew disease of spinach, caused by Peronospora effusa, is managed in conventional fields by a combination of host resistance and scheduled fungicide applications. Fungicides are currently applied to prevent downy mildew epidemics regardless of the infection status of spinach crops. A more streamlined approach would be to develop methods to target either latent infections for fungicide application in conventional production systems or to hasten harvest in organic production. In this study, conventional polymerase chain reaction (PCR) was applied to detect P. effusa DNA in symptomless spinach leaves in three spatially and temporally separated field plots, each containing four 2-m beds, 35 m in length. Spinach leaves were sampled weekly at 3-m intervals at 48 locations throughout each plot. Initial samples were asymptomatic and yet PCR enabled detection of P. effusa DNA extracted from sampled spinach leaves. Detection of latent downy mildew infection in spinach leaves was confirmed by PCR as early as 7 days prior to symptom development. The limit of pathogen DNA detection in spinach leaves was calculated at 10 pg using the conventional PCR approach. Quantitative PCR with TaqMan methodology revealed the presence of inhibitors from spinach leaf DNA extracts and affected amplification efficiencies, but not when diluted, enabling detection of P. effusa DNA at a concentration of <0.1 pg. In conclusion, detection of latent infections may enable management decisions for earlier-than-normal harvest of infected, symptomless organic crops, and for timing fungicide applications on symptomless plants in conventional production.