Rewiring mitogen-activated protein kinase cascade by positive feedback confers potato blight resistance

Abstract

Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P. infestans. The pathogen attack provoked defense-related MAPK activation followed by induction of NADPH oxidase gene expression, which is implicated in reactive oxygen species production, and resulted in hypersensitive response-like phenotype. We propose that enhancing disease resistance through altered regulation of plant defense mechanisms should be more durable and publicly acceptable than engineering overexpression of antimicrobial proteins.

Figure 1.

A scheme of stimulus-responsive isoprenoid biosynthesis in solanaceous plants. Sesquiterpene cyclase is a key branch enzyme of isoprenoid pathway for the synthesis of sesquiterpenoid phytoalexins. Vetispiradiene synthase, which catalyzes farnesyl diphosphate into vetispiradiene (1, 2, 3b), produces a precursor of lubimin and rishitin in potato and H. muticus. 5-epi-Aristolochene synthase is a key enzyme for capsidiol production in tobacco and pepper (1, 2, 3a). Wound-induced sterol and steroid glycoalkaloid syntheses are suppressed in favor of sesquiterpenoid phytoalexin synthesis during expression of the HR.

Figure 2.

PVS3 gene is activated by virulent and avirulent isolates of P. infestans in potato tubers and leaves. A, A schematic representation of amino cid sequence alignment between tobacco (TEAS), potato (PVS1, 3, and 4), H. muticus (HVS), and pepper (PEAS). Solid vertical bars correspond to intron positions within the tobacco, potato, H. muticus, and pepper genes. Numbers within the boxes indicate the number of amino acids encoded by exons. Aristlochene-specific domains and vetispiradiene-specific domains are shown as gray and black boxes, respectively. Percentages refer to identity scores between the indicated domains, and DDXXD (or DDXX) refers to Asp-rich (and known as the substrate-binding site) residues. Adapted from Back and Chappell (1995). B, The PVS3 gene is induced in both incompatible (Incomp.) and compatible (Comp.) interactions of P. infestans, but not by wounding (Wound.). RNA was extracted from tubers and leaves after the inoculation with virulent (race 1.2.3.4) and avirulent (race 0) isolates of P. infestans or wounding with Carborundum, and used for RT-PCR. Member-specific primers of PVS1 to 4 were used for PCR. RT-PCR products from leaves were separated on an agarose gel and blotted onto nylon membranes. The membranes were hybridized with each ³²P-labeled PCR product as probes. Lane TI shows RT-PCR products of RNA isolated from potato tubers in incompatible interaction as a positive control.

Figure 3.

Expression profile of GUS gene under the control of PVS3 promoter in the transgenic potato tubers and leaves. Transgenic potato tubers harboring PVS3::GUS were inoculated with the virulent P. infestans (A), mock (B), and E. coli (C). The transgenic potato leaves were treated with wounding (D) or inoculated with A. solani (E), P. infestans (F), and A. alternata Japanese pear pathotype (G). Agrobacterium-carrying vector (H), 35S::Avr9/Cf-9 (I), or 35S::StMEK1^DD (J) was infiltrated into transgenic potato leaves. Genes for Avr9/Cf-9 and StMEK1^DD were driven by the 35S promoter of Cauliflower mosaic virus. Tubers and leaves were stained with GUS staining solution 9 h (A), 24 h (F), or 48 h (B–E, and G–J) after the treatments. Scale bars = 10 μm.

Figure 4.

PVS3 promoter is controlled by both SIPK and WIPK. A, Scheme of reporter and effector plasmids used in transient assays. The reporter plasmid PVS3 promoter fragment (positions −2,334 to +30) was translationally fused to the GUS gene containing intron (GUSint). The effector plasmid estradiol-inducible promoter was fused to the StMEK1^DD gene. B, Half leaf of gene-silenced N. benthamiana was coinfiltrated with a mixture of Agrobacterium harboring PVS3::GUSint (reporter) or pER8::StMEK1^DD (effector). Estradiol (10 μm) was injected 48 h after agroinfiltration to activate the effector. GUS activities driven by PVS3 promoter in response to StMEK1^DD were measured in gene-silenced N. benthamiana by PVX (PVX-control [PVX], WIPK [PVX:W], SIPK [PVX:S], SIPK/WIPK [PVX:S/W]) 24 h after estradiol injection. Results are presented as relative values calibrated by GUS activity, which were measured by fluorometric quantitation of 4-MU, under the control of 35S promoter in another half leaf. The PVX-control with StMEK1^DD was arbitrarily assigned as 100% value against which all other values were plotted. C, Expression profiles of EAS in the gene-silenced N. benthamiana. Total RNA was isolated from gene-silenced N. benthamiana leaves after infiltration with Agrobacterium harboring 35S::StMEK1^DD, and transcript levels of WIPK, SIPK, and EAS were determined by RNA gel-blot hybridization.

Figure 5.

Deletion analysis of the PVS3 promoter in response to StMEK1^DD and INF1 in N. benthamiana leaves. A series of 5′-deleted PVS3 promoter fragments was translationally fused to the GUSint reporter gene. The number indicates the distance from the PVS3 translation start site. To induce the expression of StMEK1^DD, 20 μm estradiol was injected into the leaves 48 h after a mixture of Agrobacterium cultures containing PVS3::GUSint (reporter) and pER8::StMEK1^DD (effector) was coinfiltrated. Ten micrograms mL⁻¹ INF1 was injected into leaves 48 h after a mixture of Agrobacterium cultures containing PVS3::GUSint was infiltrated. GUS activities were determined 24 h after the treatments and measured by fluorometric quantitation of 4-MU. Each value and bar represents the mean of three independent experiments and sd from the mean, respectively.

Figure 6.

A 51-kD MAPK is activated during incompatible and compatible P. infestans-potato interactions in potato leaves. A 51-kD MAPK activity, which was identified as StMPK1 (AB062138), was indicated by in-gel kinase assay using MBP as a substrate. The same samples were stained with Coomassie Brilliant Blue, and the bands corresponding to ribulose-1,5-bisphosphate carboxylase large subunit (RBCL) are shown.

Figure 7.

Transgenic potato plants harboring PVS3::StMEK1^DD show resistance to P. infestans and A. solani. A, The transgenic plants developed normally. B, The transgenic tubers developed normally. C, Eleven days after inoculation of wild-type and transgenic leaves with virulent P. infestans. D, HR-like cell death was observed under the microscope 24 h after inoculation with virulent P. infestans in transgenic leaves. E, H₂O₂ accumulation visualized by DAB was observed under the microscope 12 h after the inoculation. F, Three days after inoculation of wild-type and transgenic potato tubers with virulent P. infestans. G, Six days after inoculation of wild-type and transgenic leaves with A. solani. H, H₂O₂ accumulation was observed 24 h after the inoculation. Scale bars = 10 μm.

Figure 8.

Transgenic potato plants indicate elevation of MAPK activity and up-regulation of defense-related genes during compatible P. infestans-potato interactions. Total RNA and proteins were isolated from potato leaves after the inoculation with virulent P. infestans at indicated times and used for RT-PCR (A), in-gel kinase assay (B), or RNA gel-blot analyses (C). For RT-PCR, 28, 40, and 30 amplification cycles were applied with specific primers for StMEK1^DD transgene, endogenous PVS3, and constitutively expressed EF-1α, respectively. The MAPK activity was assayed with in-gel kinase assay using MBP as a substrate. The same samples were stained with Coomassie Brilliant Blue, and the bands corresponding to ribulose-1,5-bisphosphate carboxylase large subunit (RBCL) are shown. The transcript levels of defense-related genes were determined by sequential probing with cDNA probes indicated on the left side of the sections.

Figure 9.

Schematic representation of mechanism of immune responses in transgenic potato plants. PAMPs derived from pathogenic microorganisms activate endogenous MAPKs (StMPK1 and StWIPK) to some extent. Following the activation of MAPKs, StMEK1^DD driven by PVS3 promoter is expressed. The expression of StrbohC and StrbohD by MAPKs produces H₂O₂ that triggers activation of MAPKs. These create positive feedback genetic circuits resulting in long-lasting activation of MAPKs.