Pangenome characterization and analysis of the NAC gene family reveals genes for Sclerotinia sclerotiorum resistance in sunflower (Helianthus annuus).

BACKGROUND: Sunflower (Helianthus annuus) is one of the most important economic crops in oilseed production worldwide. The different cultivars exhibit variability in their resistance genes. The NAC transcription factor (TF) family plays diverse roles in plant development and stress responses. With the completion of the H. annuus genome sequence, the entire complement of genes coding for NACs has been identified. However, the reference genome of a single individual cannot cover all the genetic information of the species. RESULTS: Considering only a single reference genome to study gene families will miss many meaningful genes. A pangenome-wide survey and characterization of the NAC genes in sunflower species were conducted. In total, 139 HaNAC genes are identified, of which 114 are core and 25 are variable. Phylogenetic analysis of sunflower NAC proteins categorizes these proteins into 16 subgroups. 138 HaNACs are randomly distributed on 17 chromosomes. SNP-based haplotype analysis shows haplotype diversity of the HaNAC genes in wild accessions is richer than in landraces and modern cultivars. Ten HaNAC genes in the basal stalk rot (BSR) resistance quantitative trait loci (QTL) are found. A total of 26 HaNAC genes are differentially expressed in response to Sclerotinia head rot (SHR). A total of 137 HaNAC genes are annotated in Gene Ontology (GO) and are classified into 24 functional groups. GO functional enrichment analysis reveals that HaNAC genes are involved in various functions of the biological process. CONCLUSIONS: We identified NAC genes in H. annuus (HaNAC) on a pangenome-wide scale and analyzed S. sclerotiorum resistance-related NACs. This study provided a theoretical basis for further genomic improvement targeting resistance-related NAC genes in sunflowers.

XYLEM CYSTEINE PEPTIDASE 1 and its inhibitor CYSTATIN 6 regulate pattern-triggered immunity by modulating the stability of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D.

Plants produce a burst of reactive oxygen species (ROS) after pathogen infection to successfully activate immune responses. During pattern-triggered immunity (PTI), ROS are primarily generated by the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). RBOHD is degraded in the resting state to avoid inappropriate ROS production; however, the enzyme mediating RBOHD degradation and how to prevent RBOHD degradation after pathogen infection is unclear. In this study, we identified an Arabidopsis (Arabidopsis thaliana) vacuole-localized papain-like cysteine protease, XYLEM CYSTEINE PEPTIDASE 1 (XCP1), and its inhibitor CYSTATIN 6 (CYS6). Pathogen-associated molecular pattern-induced ROS burst and resistance were enhanced in the xcp1 mutant but were compromised in the cys6 mutant, indicating that XCP1 and CYS6 oppositely regulate PTI responses. Genetic and biochemical analyses revealed that CYS6 interacts with XCP1 and depends on XCP1 to enhance PTI. Further experiments showed that XCP1 interacts with RBOHD and accelerates RBOHD degradation in a vacuole-mediated manner. CYS6 inhibited the protease activity of XCP1 toward RBOHD, which is critical for RBOHD accumulation upon pathogen infection. As CYS6, XCP1, and RBOHD are conserved in all plant species tested, our findings suggest the existence of a conserved strategy to precisely regulate ROS production under different conditions by modulating the stability of RBOHD.

A simple and efficient strategy to produce transgene-free gene edited plants in one generation using paraquat resistant 1 as a selection marker.

Introduction: DNA integration is a key factor limiting the marketing of CRISPR/Cas9-mediated gene edited crops. Several strategies have been established to obtain transgene-free gene edited plants; however, these strategies are usually time-consuming, technically difficult, providing low mutagenesis efficiency, and/or including a narrow host range. Method: To overcome such issues, we established a paraquat resistant 1 (PAR1)-based positive screening (PARS) strategy, which achieved efficient screening of transgene-free gene edited plants. Results: With PARS, the screening efficiency of mutant increased by 2.81-fold on average, and approximately 10% of T1 plants selected via PARS were transgenefree. Moreover, heritable transgene-free mutations at target loci were identified in the T1 generation. Discussion: Based on the previous reports and our data, we know that paraquat is toxic to all green plants, PAR1 is conserved among all plant species tested, and the transient expression of Cas9 editor can produce transgene-free gene edited plants. Thus, we assume that the PARS strategy established here has the potential to be widely used to screen transgene-free mutants in various crops using diverse CRISPR/Cas9 delivery approaches.

The THO/TREX Complex Active in Alternative Splicing Mediates Plant Responses to Salicylic Acid and Jasmonic Acid.

Salicylic acid (SA) and jasmonic acid (JA) are essential plant immune hormones, which could induce plant resistance to multiple pathogens. However, whether common components are employed by both SA and JA to induce defense is largely unknown. In this study, we found that the enhanced disease susceptibility 8 (EDS8) mutant was compromised in plant defenses to hemibiotrophic pathogen Pseudomonas syringae pv. maculicola ES4326 and necrotrophic pathogen Botrytis cinerea, and was deficient in plant responses to both SA and JA. The EDS8 was identified to be THO1, which encodes a subunit of the THO/TREX complex, by using mapping-by-sequencing. To check whether the EDS8 itself or the THO/TREX complex mediates SA and JA signaling, the mutant of another subunit of the THO/TREX complex, THO3, was tested. THO3 mutation reduced both SA and JA induced defenses, indicating that the THO/TREX complex is critical for plant responses to these two hormones. We further proved that the THO/TREX interacting protein SERRATE, a factor regulating alternative splicing (AS), was involved in plant responses to SA and JA. Thus, the AS events in the eds8 mutant after SA or JA treatment were determined, and we found that the SA and JA induced different alternative splicing events were majorly modulated by EDS8. In summary, our study proves that the THO/TREX complex active in AS is involved in both SA and JA induced plant defenses.

GLABRA2-based selection efficiently enriches Cas9-generated nonchimeric mutants in the T1 generation.

The CRISPR/Cas9 system is a widely used tool for genome editing in plants. In Arabidopsis (Arabidopsis thaliana), egg cell-specific promoters driving Cas9 expression have been applied to reduce the proportion of T1 transformants that are chimeras; however, this approach generally leads to relatively low mutagenesis rates. In this study, a GLABRA2 mutation-based visible selection (GBVS) system was established to enrich nonchimeric mutants among T1 plants generated by an egg cell-specific CRISPR/Cas9 system. GBVS generally enhanced mutation screening, increasing the frequency by 2.58- to 7.50-fold, and 25%-48.15% of T1 plants selected through the GBVS system were homozygous or biallelic mutants, which was 1.71- to 7.86-fold higher than the percentage selected using the original system. The mutant phenotypes of T2 plants were not obviously affected by the glabrous background for all four target genes used in this study. Additionally, the nonchimeric pyrabactin resistance 1 (PYR1)/PYR1-like 1 (PYL1) and PYL2 triple mutant pyr1/pyl1/pyl2 could be obtained in the T1 generation with a ratio of 26.67% when GBVS was applied. Collectively, our results show that compared with the known CRISPR/Cas9 systems, the GBVS system described here saves more time and labor when used for the obtainment of homozygous or biallelic monogenic mutants and nonchimeric polygenic mutants in Arabidopsis.

FgPex3, a Peroxisome Biogenesis Factor, Is Involved in Regulating Vegetative Growth, Conidiation, Sexual Development, and Virulence in Fusarium graminearum.

Peroxisomes are involved in a wide range of important cellular functions. Here, the role of the peroxisomal membrane protein PEX3 in the plant-pathogen and mycotoxin producer Fusarium graminearum was studied using knock-out and complemented strains. To fluorescently label peroxisomes' punctate structures, GFP and RFP fusions with the PTS1 and PTS2 localization signal were transformed into the wild type PH-1 and ΔFgPex3 knock-out strains. The GFP and RFP transformants in the ΔFgPex3 background showed a diffuse fluorescence pattern across the cytoplasm suggesting the absence of mature peroxisomes. The ΔFgPex3 strain showed a minor, non-significant reduction in growth on various sugar carbon sources. In contrast, deletion of FgPex3 affected fatty acid ß-oxidation in F. graminearum and significantly reduced the utilization of fatty acids. Furthermore, the ΔFgPex3 mutant was sensitive to osmotic stressors as well as to cell wall-damaging agents. Reactive oxygen species (ROS) levels in the mutant had increased significantly, which may be linked to the reduced longevity of cultured strains. The mutant also showed reduced production of conidiospores, while sexual reproduction was completely impaired. The pathogenicity of ΔFgPex3, especially during the process of systemic infection, was strongly reduced on both tomato and on wheat, while to production of deoxynivalenol (DON), an important factor for virulence, appeared to be unaffected.

The Fusarium graminearum Histone Acetyltransferases Are Important for Morphogenesis, DON Biosynthesis, and Pathogenicity.

Post-translational modifications of chromatin structure by histone acetyltransferase (HATs) play a central role in the regulation of gene expression and various biological processes in eukaryotes. Although HAT genes have been studied in many fungi, few of them have been functionally characterized. In this study, we identified and characterized four putative HATs (FgGCN5, FgRTT109, FgSAS2, FgSAS3) in the plant pathogenic ascomycete Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. We replaced the genes and all mutant strains showed reduced growth of F. graminearum. The ΔFgSAS3 and ΔFgGCN5 mutant increased sensitivity to oxidative and osmotic stresses. Additionally, ΔFgSAS3 showed reduced conidia sporulation and perithecium formation. Mutant ΔFgGCN5 was unable to generate any conidia and lost its ability to form perithecia. Our data showed also that FgSAS3 and FgGCN5 are pathogenicity factors required for infecting wheat heads as well as tomato fruits. Importantly, almost no Deoxynivalenol (DON) was produced either in ΔFgSAS3 or ΔFgGCN5 mutants, which was consistent with a significant downregulation of TRI genes expression. Furthermore, we discovered for the first time that FgSAS3 is indispensable for the acetylation of histone site H3K4, while FgGCN5 is essential for the acetylation of H3K9, H3K18, and H3K27. H3K14 can be completely acetylated when FgSAS3 and FgGCN5 were both present. The RNA-seq analyses of the two mutant strains provide insight into their functions in development and metabolism. Results from this study clarify the functional divergence of HATs in F. graminearum, and may provide novel targeted strategies to control secondary metabolite expression and infections of F. graminearum.

Host and Cropping System Shape the Fusarium Population: 3ADON-Producers Are Ubiquitous in Wheat Whereas NIV-Producers Are More Prevalent in Rice.

In recent years, Fusarium head blight (FHB) outbreaks have occurred much more frequently in China. The reduction of burning of the preceding crop residues is suggested to contribute to more severe epidemics as it may increase the initial inoculum. In this study, a large number of Fusarium isolates was collected from blighted wheat spikes as well as from rice stubble with perithecia originating from nine sampling sites in five provinces in Southern China. Fusarium asiaticum dominated both wheat and rice populations, although rice populations showed a higher species diversity. Chemotype analysis showed that rice is the preferred niche for NIV mycotoxin producers that were shown to be less virulent on wheat. In contrast, 3ADON producers are more prevalent on wheat and in wheat producing areas. The 3ADON producers were shown to be more virulent on wheat, revealing the selection pressure of wheat on 3ADON producers. For the first time, members of the Incarnatum-clade of FusariumIncarnatum-Equiseti Species Complex (FIESC) were found to reproduce sexually on rice stubble. The pathogenicity of FIESC isolates on wheat proved very low and this may cause the apparent absence of this species in the main wheat producing provinces. This is the first report of the Fusarium population structure including rice stubble as well as a direct comparison with the population on wheat heads in the same fields. Our results confirm that the perithecia on rice stubble are the primary inoculum of FHB on wheat and that cropping systems affect the local Fusarium population.

Corrigendum: Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China.

[This corrects the article on p. 76 in vol. 8, PMID: 28197157.].

Corrigendum: Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China.

[This corrects the article on p. 76 in vol. 8, PMID: 28197157.].

Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China.

Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 102 CFU/ml of bacterial cells per 25 µl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient.

Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola.

A rapid LAMP (loop-mediated isothermal amplification) detection method was developed on the basis of the ITS sequence of P. viticola, the major causal agent of grape downy mildew. Among the 38 fungal and oomycete species tested, DNA isolated exclusively from P. viticola resulted in a specific product after LAMP amplification. This assay had high sensitivity and was able to detect the presence of less than 33 fg of genomic DNA per 25-µL reaction within 30 min. The infected leaves may produce sporangia that serve as a secondary inoculum. The developed LAMP assay is efficient for estimating the latent infection of grape leaves by P. viticola. When combined with the rapid and simple DNA extraction method, this assay's total detection time is shortened to approximately one hour; therefore it is suitable for on-site detection of latent infection in the field. The sporangia levels in the air are strongly associated with disease severity. The LAMP method was also demonstrated to be able to estimate the level of sporangia released in the air in a certain period. This assay should make disease forecasting more accurate and rapid and should be helpful in decision-making regarding the control of grape downy mildew.