A Single Nucleotide Mutation in Adenylate Cyclase Affects Vegetative Growth, Sclerotial Formation and Virulence of Botrytis cinerea.

Botrytis cinerea is a pathogenic fungus that causes gray mold disease in a broad range of crops. The high intraspecific variability of B. cinerea makes control of this fungus very difficult. Here, we isolated a variant B05.10M strain from wild-type B05.10. The B05.10M strain showed serious defects in mycelial growth, spore and sclerotia production, and virulence. Using whole-genome resequencing and site-directed mutagenesis, a single nucleotide mutation in the adenylate cyclase (BAC) gene that results in an amino acid residue (from serine to proline, S1407P) was shown to be the cause of various defects in the B05.10M strain. When we further investigated the effect of S1407 on BAC function, the S1407P mutation in bac showed decreased accumulation of intracellular cyclic AMP (cAMP), and the growth defect could be partially restored by exogenous cAMP, indicating that the S1407P mutation reduced the enzyme activity of BAC. Moreover, the S1407P mutation exhibited decreased spore germination rate and infection cushion formation, and increased sensitivity to cell wall stress, which closely related to fungal development and virulence. Taken together, our study indicates that the S1407 site of bac plays an important role in vegetative growth, sclerotial formation, conidiation and virulence in B. cinerea.

EDR1 physically interacts with MKK4/MKK5 and negatively regulates a MAP kinase cascade to modulate plant innate immunity.

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity.

BR-SIGNALING KINASE1 physically associates with FLAGELLIN SENSING2 and regulates plant innate immunity in Arabidopsis.

Pathogen-associated molecular pattern (PAMP)-trigged immunity (PTI) is the first defensive line of plant innate immunity and is mediated by pattern recognition receptors. Here, we show that a mutation in BR-SIGNALING KINASE1 (BSK1), a substrate of the brassinosteroid (BR) receptor BRASSINOSTEROID INSENSITIVE1, suppressed the powdery mildew resistance caused by a mutation in ENHANCED DISEASE RESISTANCE2, which negatively regulates powdery mildew resistance and programmed cell death, in Arabidopsis thaliana. A loss-of-function bsk1 mutant displayed enhanced susceptibility to virulent and avirulent pathogens, including Golovinomyces cichoracearum, Pseudomonas syringae, and Hyaloperonospora arabidopsidis. The bsk1 mutant also accumulated lower levels of salicylic acid upon infection with G. cichoracearum and P. syringae. BSK1 belongs to a receptor-like cytoplasmic kinase family and displays kinase activity in vitro; this kinase activity is required for its function. BSK1 physically associates with the PAMP receptor FLAGELLIN SENSING2 and is required for a subset of flg22-induced responses, including the reactive oxygen burst, but not for mitogen-activated protein kinase activation. Our data demonstrate that BSK1 is involved in positive regulation of PTI. Together with previous findings, our work indicates that BSK1 represents a key component directly involved in both BR signaling and plant immunity.

Haplotype-based phylogenetic analysis and population genomics uncover the origin and domestication of sweetpotato.

The hexaploid sweetpotato (Ipomoea batatas) is one of the most important root crops worldwide. However, its genetic origin remains controversial, and its domestication history remains unknown. In this study, we used a range of genetic evidence and a newly developed haplotype-based phylogenetic analysis to identify two probable progenitors of sweetpotato. The diploid progenitor was likely closely related to Ipomoea aequatoriensis and contributed the B1 subgenome, IbT-DNA2, and the lineage 1 type of chloroplast genome to sweetpotato. The tetraploid progenitor of sweetpotato was most likely I. batatas 4x, which donated the B2 subgenome, IbT-DNA1, and the lineage 2 type of chloroplast genome. Sweetpotato most likely originated from reciprocal crosses between the diploid and tetraploid progenitors, followed by a subsequent whole-genome duplication. In addition, we detected biased gene exchanges between the subgenomes; the rate of B1 to B2 subgenome conversions was nearly three times higher than that of B2 to B1 subgenome conversions. Our analyses revealed that genes involved in storage root formation, maintenance of genome stability, biotic resistance, sugar transport, and potassium uptake were selected during the speciation and domestication of sweetpotato. This study sheds light on the evolution of sweetpotato and paves the way for improvement of this crop.

RPN1a, a 26S proteasome subunit, is required for innate immunity in Arabidopsis.

Accumulating evidence shows that proper degradation of proteins that affect defense responses in a positive or negative manner is critical in plant immunity. However, the role of plant degradation systems such as the 26S proteasome in plant immunity is not well understood. Loss-of-function mutations in EDR2 (ENHANCED DISEASE RESISTANCE 2) lead to increased resistance to the adapted biotrophic powdery mildew pathogen Golovinomyces cichoracearum. To study the molecular interactions between powdery mildew pathogen and Arabidopsis, we performed a screen for suppressors of edr2 and found that mutation in the gene that encodes RPN1a, a subunit of the 26S proteasome, suppressed edr2-associated disease resistance phenotypes. In addition, RPN1a is required for edr1- and pmr4-mediated powdery mildew resistance and mildew-induced cell death. Furthermore, we show that rpn1a displayed enhanced susceptibility to the fungal pathogen G. cichoracearum and to virulent and avirulent bacterial Pto DC3000 strains, which indicated that rpn1a has defects in basal defense and resistance (R) protein-mediated defense. RPN1a-GFP localizes to both the nucleus and cytoplasm. Accumulation of RPN1a is affected by salicylic acid (SA) and the rpn1a mutant has defects in SA accumulation upon Pto DC3000 infection. Further analysis revealed that two other subunits of the 26S proteasome, RPT2a and RPN8a are also involved in edr2-mediated disease resistance. Based on these results, we conclude that RPN1a is required for basal defense and R protein-mediated defense. Our data provide evidence that some subunits of the 26S proteasome are involved in innate immunity in Arabidopsis.

SR1, a calmodulin-binding transcription factor, modulates plant defense and ethylene-induced senescence by directly regulating NDR1 and EIN3.

Plant defense responses are tightly controlled by many positive and negative regulators to cope with attacks from various pathogens. Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE2 (EDR2) is a negative regulator of powdery mildew resistance, and edr2 mutants display enhanced resistance to powdery mildew (Golovinomyces cichoracearum). To identify components acting in the EDR2 pathway, we screened for edr2 suppressors and identified a gain-of-function mutation in SIGNAL RESPONSIVE1 (SR1), which encodes a calmodulin-binding transcription activator. The sr1-4D gain-of-function mutation suppresses all edr2-associated phenotypes, including powdery mildew resistance, mildew-induced cell death, and ethylene-induced senescence. The sr1-4D single mutant is more susceptible to a Pseudomonas syringae pv tomato DC3000 virulent strain and to avirulent strains carrying avrRpt2 or avrRPS4 than the wild type. We show that SR1 directly binds to the promoter region of NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), a key component in RESISTANCE TO PSEUDOMONAS SYRINGAE2-mediated plant immunity. Also, the ndr1 mutation suppresses the sr1-1 null allele, which shows enhanced resistance to both P. syringae pv tomato DC3000 avrRpt2 and G. cichoracearum. In addition, we show that SR1 regulates ethylene-induced senescence by directly binding to the ETHYLENE INSENSITIVE3 (EIN3) promoter region in vivo. Enhanced ethylene-induced senescence in sr1-1 is suppressed by ein3. Our data indicate that SR1 plays an important role in plant immunity and ethylene signaling by directly regulating NDR1 and EIN3.

A New V361A Mutation in Amaranthus palmeri PPX2 Associated with PPO-Inhibiting Herbicide Resistance.

Weeds resistant to PPO-inhibiting herbicides threaten the profitability of crop producers relying on this chemistry. In Amaranthus palmeri, mutations at G210 (∆G210) and R128 (R128G/M) of the PPX2 gene were reported to confer PPO-inhibitor resistance. Here, A. palmeri samples from nine states in America, having survived a field application of a PPO-inhibitor, were genotyped to determine the prevalence of these mutations. Less than 5% of the 1828 A. palmeri plants screened contained the ∆G210 mutation. Of the plants lacking ∆G210, a R128 substitution was only found in a single plant. An A. palmeri population from Alabama without mutations at G210 or R128 had a resistance ratio of 3.1 to 3.5 for fomesafen. Of the candidate PPX2 mutations identified in this population, only V361A conferred resistance to lactofen and fomesafen in a transformed bacterial strain. This is the first report of the V361A substitution of PPX2 conferred PPO-inhibiting herbicide resistance in any plant species. Future molecular screens of PPO-inhibitor resistance in A. palmeri and other species should encompass the V361A mutation of PPX2 to avoid false-negative results.

FERONIA coordinates plant growth and salt tolerance via the phosphorylation of phyB.

Phosphorylation modification is required for the modulation of phytochrome B (phyB) thermal reversion, but the kinase(s) that phosphorylate(s) phyB and the biological significance of the phosphorylation are still unknown. Here we report that FERONIA (FER) phosphorylates phyB to regulate plant growth and salt tolerance, and the phosphorylation not only regulates dark-triggered photobody dissociation but also modulates phyB protein abundance in the nucleus. Further analysis indicates that phosphorylation of phyB by FER is sufficient to accelerate the conversion of phyB from the active form (Pfr) to the inactive form (Pr). Under salt stress, FER kinase activity is inhibited, leading to delayed photobody dissociation and increased phyB protein abundance in the nucleus. Our data also show that phyB mutation or overexpression of PIF5 attenuates growth inhibition and promotes plant survival under salt stress. Together, our study not only reveals a kinase that controls phyB turnover via a signature of phosphorylation, but also provides mechanistic insights into the role of the FER-phyB module in coordinating plant growth and stress tolerance.

Exploring and exploiting genetics and genomics for sweetpotato improvement: Status and perspectives.

Sweetpotato (Ipomoea batatas (L.) Lam.) is one of the most important root crops cultivated worldwide. Because of its adaptability, high yield potential, and nutritional value, sweetpotato has become an important food crop, particularly in developing countries. To ensure adequate crop yields to meet increasing demand, it is essential to enhance the tolerance of sweetpotato to environmental stresses and other yield-limiting factors. The highly heterozygous hexaploid genome of I. batatas complicates genetic studies and limits improvement of sweetpotato through traditional breeding. However, application of next-generation sequencing and high-throughput genotyping and phenotyping technologies to sweetpotato genetics and genomics research has provided new tools and resources for crop improvement. In this review, we discuss the genomics resources that are available for sweetpotato, including the current reference genome, databases, and available bioinformatics tools. We systematically review the current state of knowledge on the polyploid genetics of sweetpotato, including studies of its origin and germplasm diversity and the associated mapping of important agricultural traits. We then outline the conventional and molecular breeding approaches that have been applied to sweetpotato. Finally, we discuss future goals for genetic studies of sweetpotato and crop improvement via breeding in combination with state-of-the-art multi-omics approaches such as genomic selection and gene editing. These approaches will advance and accelerate genetic improvement of this important root crop and facilitate its sustainable global production.

CgSCD1 Is Essential for Melanin Biosynthesis and Pathogenicity of Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides, an important phytopathogenic fungus, mainly infects tropical fruits and results in serious anthracnose. Previous studies have shown that melanin biosynthesis inhibitor can inhibit the melanization of the appressoria of Magnaporthe grisea and Colletotrichum orbiculare, resulting in limited infection of the hosts. In this study, we identified and characterized a scytalone dehydratase gene (CgSCD1) from C. gloeosporioides which is involved in melanin synthesis. The CgSCD1 gene deletion mutant ΔCgscd1 was obtained using homologous recombination. The ΔCgscd1 mutant showed no melanin accumulation on appressoria formation and vegetative hyphae. Furthermore, the virulence of ΔCgscd1 was significantly reduced in comparison with the wild-type (WT) strain. Further investigations showed that the growth rate as well as germination and appressorium formation of ΔCgscd1 displayed no difference compared to the wild-type and complemented transformant Cgscd1com strains. Furthermore, we found that the appressorial turgor pressure in the ΔCgscd1 mutant showed no difference compared to that in the WT and Cgscd1com strains in the incipient cytorrhysis experiment. However, fewer infectious hyphae of ΔCgscd1 were observed in the penetration experiments, suggesting that the penetration ability of nonpigmented appressoria was partially impaired. In conclusion, we identified the CgSCD1 gene, which is involved in melanin synthesis and pathogenicity, and found that the melanization defect did not affect appressorial turgor pressure in C. gloeosporioides.

The Photoreceptor Components FaWC1 and FaWC2 of Fusarium asiaticum Cooperatively Regulate Light Responses but Play Independent Roles in Virulence Expression.

Fusarium asiaticum belongs to one of the phylogenetical subgroups of the F. graminearum species complex and is epidemically predominant in the East Asia area. The life cycle of F. asiaticum is significantly regulated by light. In this study, the fungal blue light receptor white collar complex (WCC), including FaWC1 and FaWC2, were characterized in F. asiaticum. The knockout mutants ΔFawc1 and ΔFawc2 were generated by replacing the target genes via homologous recombination events. The two mutants showed similar defects in light-induced carotenoid biosynthesis, UV-C resistance, sexual fruiting body development, and the expression of the light-responsive marker genes, while in contrast, all these light responses were characteristics in wild-type (WT) and their complementation strains, indicating that FaWC1 and FaWC2 are involved in the light sensing of F. asiaticum. Unexpectedly, however, the functions of Fawc1 and Fawc2 diverged in regulating virulence, as the ΔFawc1 was avirulent to the tested host plant materials, but ΔFawc2 was equivalent to WT in virulence. Moreover, functional analysis of FaWC1 by partial disruption revealed that its light-oxygen-voltage (LOV) domain was required for light sensing but dispensable for virulence, and its Zinc-finger domain was required for virulence expression but not for light signal transduction. Collectively, these results suggest that the conserved fungal blue light receptor WCC not only endows F. asiaticum with light-sensing ability to achieve adaptation to environment, but it also regulates virulence expression by the individual component FaWC1 in a light-independent manner, and the latter function opens a way for investigating the pathogenicity mechanisms of this important crop disease agent.

Confirmation of herbicide resistance mutations Trp574Leu, ΔG210, and EPSPS gene amplification and control of multiple herbicide-resistant Palmer amaranth (Amaranthus palmeri) with chlorimuron-ethyl, fomesafen, and glyphosate.

Herbicide-resistant weeds, especially Palmer amaranth (Amaranthus palmeri S. Watson), are problematic in row-crop producing areas of the United States. The objectives of this study were to determine if chlorimuron-ethyl, fomesafen, and glyphosate applied separately and in mixtures control A. palmeri and confirm the presence of various genotypes surviving two- and three-way herbicide mixtures. Fifteen percent of A. palmeri treated with the three-way herbicide mixture survived. Mixing fomesafen with chlorimuron-ethyl or fomesafen with glyphosate to create a two-way mixture reduced A. palmeri survival 22 to 24% and 60 to 62% more than glyphosate and chlorimuron-ethyl alone, respectively. Previously characterized mutations associated with A. palmeri survival to chlorimuron-ethyl, fomesafen, and glyphosate Trp574Leu, a missing glycine codon at position 210 of the PPX2L gene (ΔG210), and 5-enolpyruvylshikimate-3-phosphase synthase (EPSPS) gene amplification; respectively, were present in surviving plants. However, 37% of plants treated with chlorimuron-ethyl did not contain heterozygous or homozygous alleles for the Trp574Leu mutation, suggesting alternative genotypes contributed to plant survival. All surviving A. palmeri treated with fomesafen or glyphosate possessed genotypes previously documented to confer resistance. Indiana soybean [Glycine max (L.) Merr] fields infested with A. palmeri possessed diverse genotypes and herbicide surviving plants are likely to produce seed and spread if alternative control measures are not implemented.

Investigating target-site resistance mechanism to the PPO-inhibiting herbicide fomesafen in waterhemp and interspecific hybridization of Amaranthus species using next generation sequencing.

BACKGROUND: Waterhemp (Amaranthus tuberculatus (Moq.) J. D. Sauer) is one of the most pernicious weeds in cropping systems of the USA due to its evolved resistance against several herbicide sites-of-action, including protoporphyrinogen oxidase inhibitors (PPO-R). Currently, the only source of PPO-R documented in waterhemp is ΔG210 of PPX2. Gene flow may not only lead to a transfer of herbicide-resistant alleles, but also produce a hybrid genotype more competitively fit than one or both parents. However, investigating gene flow of Amaranthus species has been of interest in the past two decades with limited evidence. RESULTS: Here, a high-throughput MiSeq amplicon sequencing method was used to investigate alterations of the PPX2 gene in 146 PPO-R waterhemp populations across five Midwest states of the USA. Five R128 codons of PPX2, novel to waterhemp, were found including AGG (R), GGA (G), GGG (G), AAA (K) and ATA (I). R128G, R128I, and R128K were found in 11, 3, and 2 populations, respectively. R128G and R128I, but not R128K, conferred fomesafen resistance in a bacterial system. Sequence alignment of the R128 region of PPX2 identified a tumble pigweed (Amaranthus albus)-type and Palmer amaranth (Amaranthus palmeri)-type PPX2 allele to be present and widespread in the surveyed waterhemp populations, thus providing strong evidence of gene flow between Amaranthus species. CONCLUSION: Using a next-generation sequencing method, we identified two PPO target-site mutations R128G/I novel to waterhemp and provided evidence of gene flow of Amaranthus species in a large group of screened waterhemp populations from five Midwest states of the USA. © 2019 Society of Chemical Industry.

Differential antioxidant enzyme activity in rapid-response glyphosate-resistant Ambrosia trifida.

BACKGROUND: The giant ragweed (Ambrosia trifida L.) rapid-response (RR) biotype exhibits a sacrificial form of glyphosate resistance whereby an oxidative burst in mature leaves results in foliage loss, while juvenile leaves remain uninjured. This work investigated the safening capacity of antioxidant enzymes in RR juvenile leaves following glyphosate treatment and examined cross tolerance to paraquat. RESULTS: Basal antioxidant enzyme activities were similar between glyphosate-susceptible (GS) and RR biotypes. Lipid peroxidation was first detected in RR mature leaves at 8 h after treatment (HAT) and by 32 HAT was 5.3 and 21.1 times greater than that in RR juvenile leaves and GS leaves, respectively. Preceding lipid peroxidation in the RR biotype at 2 and 4 HAT, the only increase in enzymatic activity was observed in ascorbate-glutathione cycle enzymes in RR juvenile leaves, particularly ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. Sensitivity to paraquat was similar between biotypes. CONCLUSION: The RR biotype is not inherently more tolerant to oxidative stress. The difference in tissue damage between RR juvenile and mature leaves following glyphosate treatment is attributable at least partially to the transient increase in antioxidant enzyme expression in juvenile leaves (0-8 HAT), but may also be attributable to lower overall RR induction in juvenile leaves compared with mature leaves. © 2018 Society of Chemical Industry.

Two new PPX2 mutations associated with resistance to PPO-inhibiting herbicides in Amaranthus palmeri.

BACKGROUND: Resistance to herbicides that inhibit protoporphyrinogen oxidase (PPO) is a widespread and growing problem for weed managers across the midwestern and midsouthern United States. In Amaranthus spp., this resistance is known to be conferred by a glycine deletion at the 210th amino acid (ΔG210) in PPO2. Preliminary analysis indicated that the ΔG210 mutation did not fully account for observed resistance to PPO inhibitors in two Amaranthus palmeri populations from Tennessee and one from Arkansas. RESULTS: Sequencing PPX2 cDNA from six resistant plants uncovered two new mutations at the R98 site (R98G and R98M), a site previously found to endow PPO-inhibitor resistance in Ambrosia artemisiifolia. Sequencing of this region from additional plants sprayed with 264 g fomesafen ha-1 showed the presence of one or both R98 mutations in a subset of the resistant plants from all three populations. No plants sensitive to fomesafen contained either mutation. A derived cleaved amplified polymorphic sequence (dCAPS) assay to test for the presence of these mutations in A. palmeri was developed. CONCLUSION: Two new mutations of PPX2 (R98G, R98M) likely confer resistance to PPO-inhibitors in A. palmeri, and can be rapidly identified using a dCAPS assay. © 2017 Society of Chemical Industry.

Suppression of edr2-mediated powdery mildew resistance, cell death and ethylene-induced senescence by mutations in ALD1 in Arabidopsis.

EDR2 is a negative regulator of the defense response and cell death in Arabidopsis. Loss-of-function of EDR2 leads to enhanced resistance to powdery mildew. To identify new components in the EDR2 signal transduction pathway, mutations that suppress edr2 resistant phenotypes were screened. Three mutants, edts5-1, edts5-2 and edts5-3 (edrtwo suppressor 5), were identified. The EDTS5 gene was identified by map-based cloning and previously was shown to encode an aminotransferase (ALD1). Therefore we renamed these three alleles ald1-10, ald1-11 and ald1-12, respectively. Mutations in ALD1 suppressed all edr2-mediated phenotypes, including powdery mildew resistance, programmed cell death and ethylene-induced senescence. Accumulation of hydrogen peroxide in edr2 was also suppressed by ald1 mutation. The expression of defense-related genes was up-regulated in the edr2 mutant, and the up-regulation of those genes in edr2 was suppressed in the edr2/ald1 double mutant. The ald1 single mutant displayed delayed ethylene-induced senescence. In addition, ald1 mutation suppressed edr1-mediated powdery mildew resistance, but could not suppress the edr1/edr2 double-mutant phenotype. These data demonstrate that ALD1 plays important roles in edr2-mediated defense responses, and senescence and revealed a crosstalk between ethylene and salicylic acid signaling mediated by ALD1 and EDR2.