Screening Universal Stress-Response Terpenoids and Their Biosynthetic Genes via Volatile and Transcriptomic Profiling in Citrus.

Volatile terpenoids accumulate in citrus and play important roles in plant defense against various stressors. However, the broad-spectrum response of terpenoid biosynthesis to ubiquitous stressors in citrus has not been comparatively investigated. In this study, volatile terpenoids were profiled under six stressors: high temperature, citrus miner, citrus red mite, citrus canker, Alternaria brown spot, and huanglongbing (HLB). Significant content changes in 15 terpenoids, including ß-ocimene, were observed in more than four of the six stressors, implying their possibly universal stress-response effects. Notably, the emission of terpenoids, including ß-caryophyllene, ß-ocimene, and nerolidol glucoside, was significantly increased by HLB in HLB-tolerant "Shatian" pomelo leaves. The upregulation of CgTPS1 and CgTPS2 and their characterization in vivo identified them as mono- or sesquiterpenoid biosynthetic genes. This study provides a foundation for determining stress resistance mechanisms in citrus and biopesticide designations for future industrial applications.

Comparative Study of the Co-Occurring Alternaria and Colletotrichum Species in the Production of Citrus Leaf Spot.

Both of the two citrus diseases, Alternaria brown spot (ABS) and Anthracnose, caused by Alternaria and Colletotrichum spp., respectively, can produce leaf lesions which are hard to differentiate. These two diseases have been confused as causal agents of brown spot for over a decade in China. In this study, citrus leaves with or without brown spot were collected from Zhaoqing, Guangdong and Wanzhou, Chongqing, and were further used for the taxonomic and functional comparisons between the co-occurring Alternaria and Colletotrichum species. In the amplicon sequencing, the average relative abundance and the composition of Alternaria, but not Colletotrichum, increased (from 0.1 to 9.9, p = 0.059; and to 0.7, p < 0.05) and significantly altered (p < 0.01) with the brown spot in Zhaoqing and Wanzhou, respectively. Two representative isolates Alternaria sp. F12A and Colletotrichum sp. F12C, from the same brown spot, were proved with different virulence and host response activation to citrus leaves. F12A caused typical symptoms of brown spot with the average spot length expanded to 5 and 6.1 cm, and also altered the citrus global gene expression 48 and 72 h after inoculation. In addition, F12A enriched the expression of genes that were most frequently involved in plant defense. In comparison, F12C caused leaf spot limited to the wounded site, and its milder activation of host response recovered 72 h after inoculation. Our study indicates that the incidence of brown spot in China is caused by Alternaria species, and the ABS should be a fungal disease of major concern on citrus.

Diversity Analysis and Function Prediction of Bacterial Communities in the Different Colored Pericarp of Citrus reticulata cv. 'Shatangju' Due to 'Candidatus Liberibacter asiaticus' Infection.

Huanglongbing (HLB), caused by the Candidatus Liberibacter spp., is the most devastating disease in the citrus industry. HLB significantly affects and alters the microbial community structure or potential function of the microbial community of leaves and roots. However, it is unknown how the microbial community structure of the pericarp with different pigments is affected by Candidatus Liberibacter asiaticus (CLas). This study identified the enriched taxa of the microbial community in the citrus pericarp with normal or abnormal pigment and determine the effects of HLB on the pericarp microbial community using 16S rRNA-seq. The alpha and beta diversity and composition of microbial communities were significantly different between normal and abnormal pigment pericarp tissues of ripe fruits infected by CLas. Firmicutes, Actinobacteriota, Bacteroidota, Acidobacteriota, and Desulfobacterota dominated the pericarp microbiota composition in WDYFs (whole dark yellow fruits) samples. The relative abundance of most genera in WDYFs was higher than 1%, such as Burkholderia, and Pelomonas. However, with the exception of the HLB pathogen, the relative abundance of most genera in the abnormal-colored pericarp samples was less than 1%. CLas decreased the relative abundance of pericarp taxonomic. The predicted function of microbial was more plentiful and functional properties in the WDYF sample, such as translation, ribosomal structure and biogenesis, amino acid transport and metabolism, energy production and conversion, and some other clusters of orthologous groups (COG) except for cell motility. The results of this study offer novel insights into understanding the composition of microbial communities of the CLas-affected citrus pericarps and contribute to the development of biological control strategies for citrus against Huanglongbing.

Integrated transcriptomic and metabolomic analyses reveal key genes controlling flavonoid biosynthesis in Citrus grandis 'Tomentosa' fruits.

As a well-recognized traditional Chinese medicine (TCM), immature fruits of Citrus grandis 'Tomentosa' (CGT) serve to cure chronic cough in humans. Specialized metabolites including flavonoids may have contribute to this curing effect. Knowledge about the molecular mechanisms underlying flavonoid biosynthesis in 'Tomentosa' fruits will, therefore, support the breeding of varieties with improved medicinal properties. Hence, we profiled the transcriptomes and metabolites of the fruits of two contrasting C. grandis varieties, namely 'Zheng-Mao' ('ZM') used in TCM production, and a locally cultivated pomelo, namely 'Guang-Qing' ('GQ'), at four developmental stages. A total of 39 flavonoids, including 14 flavanone/flavone, 5 isoflavonoids, 12 flavonols, and 6 anthocyanins, were identified, and 16 of which were quantitatively determined in the fruits of the two varieties. We found that 'ZM' fruits contain more flavonoids than 'GQ'. Specifically, rhoifolin levels were significantly higher in 'ZM' than in 'GQ'. We annotated 31,510 genes, including 1,387 previously unknown ones, via transcriptome sequencing of 'ZM' and 'GQ.' A total of 646 genes were found to be differentially expressed between 'ZM' and 'GQ' throughout at all four fruit developmental stages, indicating that they are robust expression markers for future breeding programs. Weighted gene co-expression network analysis identified 18 modules. Combined transcriptional and metabolic analysis revealed 25 genes related to flavonoid biosynthesis and 16 transcriptional regulators (MYBs, bHLHs, WD40) that may be involved in the flavonoids biosynthesis in C. grandis 'Tomentosa' fruits.

The root enrichment of bacteria is consistent across different stress-resistant plant species.

Bacteria, inhabiting around and in plant roots, confer many beneficial traits to promote plant growth and health. The secretion of root exudates modulates the nutritional state of the rhizosphere and root area, further selecting specific bacteria taxa and shaping the bacteria communities. Many studies of the rhizosphere effects have demonstrated that selection by the plant rhizosphere consistently enriches a set of bacteria taxa, and this is conserved across different plant species. Root selection effects are considered to be stronger than the rhizosphere selection effects, yet studies are limited. Here, we focus on the root selection effects across a group of 11 stress-resistant plant species. We found that the root selection consistently reduced the alpha diversity (represented by total number of observed species, Shannon's diversity, and phylogenetic diversity) and altered the structure and composition of bacteria communities. Furthermore, root selection tended to enrich for clusters of bacteria genera including Pantoea, Akkermansia, Blautia, Acinetobacter, Burkholderia-Paraburkholderia, Novosphingobium, Massilia, Pseudomonas, Chryseobacterium, and Stenotrophomonas. Our study offers some basic knowledge for understanding the microbial ecology of the plant root, and suggests that several bacteria genera are of interest for future studies.

Arsenic inhibits citric acid accumulation via downregulating vacuolar proton pump gene expression in citrus fruits.

Citric acid content is a critical quality determinant in citrus (Citrus spp.) fruits. Although arsenic (As) can effectively reduce citric acid content to improve citrus fruit quality, it can have adverse environmental effects. The discovery of nontoxic substitutes is hampered by the incomplete elucidation of the underlying mechanisms of As action in citrus fruits. Metabolic, transcriptomic, and physiological analyses were employed to investigate As action on citric acid accumulation to discover the mechanisms of As action in citrus. The enzyme activity related to citrate biosynthesis was not inhibited and the content of the involved metabolites was not reduced in As-treated fruits. However, the proton pump genes CitPH5 and CitPH1 control the vacuolar citric acid accumulation and transcription factor genes CitTT8 and CitMYB5, which regulate CitPH5 and CitPH1, were downregulated. The oxidative stress-response genes were upregulated in As-treated fruits. The reactive oxygen species (ROS) treatment also downregulated CitTT8 and CitMYB5 in juice cells. The mitochondrial ROS production rate increased in As-treated fruits. AsIII was more potent in stimulating isolated mitochondria to overproduce ROS compared to AsV. Our results indicate that the As inhibition of citric acid accumulation may be primarily due to the transcriptional downregulation of CitPH5, CitPH1, CitTT8, and CitMYB5. As-induced oxidative stress signaling may operate upstream to downregulate these acid regulator genes. Mitochondrial thiol proteins may be the principal targets of As action in citrus fruits.

Metabolic Profiling and Transcriptional Analysis of Carotenoid Accumulation in a Red-Fleshed Mutant of Pummelo (Citrus grandis).

Citrus grandis 'Tomentosa', commonly known as 'Huajuhong' pummelo (HJH), is used in traditional Chinese medicine and can moisten the lungs, resolve phlegm, and relieve coughs. A spontaneous bud mutant, named R-HJH, had a visually attractive phenotype with red albedo tissue and red juice sacs. In this study, the content and composition of carotenoids were investigated and compared between R-HJH and wild-type HJH using HPLC-MS analysis. The total carotenoids in the albedo tissue and juice sacs of R-HJH were 4.03- and 2.89-fold greater than those in HJH, respectively. The massive accumulation of carotenoids, including lycopene, ß-carotene and phytoene, led to the attractive red color of R-HJH. However, the contents of flavones, coumarins and most volatile components (mainly D-limonene and γ-terpinene) were clearly reduced in R-HJH compared with wild-type HJH. To identify the molecular basis of carotenoid accumulation in R-HJH, RNA-Seq transcriptome sequencing was performed. Among 3948 differentially expressed genes (DEGs), the increased upstream synthesis genes (phytoene synthase gene, PSY) and decreased downstream genes (ß-carotene hydroxylase gene, CHYB and carotenoid cleavage dioxygenase gene, CCD7) might be the key factors that account for the high level of carotenoids in R-HJH. These results will be beneficial for determining the molecular mechanism of carotenoid accumulation and metabolism in pummelo.

Integrative analysis of metabolome and transcriptome profiles provides insight into the fruit pericarp pigmentation disorder caused by 'Candidatus Liberibacter asiaticus' infection.

BACKGROUND: Mandarin 'Shatangju' is susceptible to Huanglongbing (HLB) and the HLB-infected fruits are small, off-flavor, and stay-green at the maturity period. To understand the relationship between pericarp color and HLB pathogen and the effect mechanism of HLB on fruit pericarp coloration, quantitative analyses of HLB bacterial pathogens and carotenoids and also the integrative analysis of metabolome and transcriptome profiles were performed in the mandarin 'Shatangju' variety with four different color fruits, whole green fruits (WGF), top-yellow and base-green fruits (TYBGF), whole light-yellow fruits (WLYF), and whole dark-yellow fruits (WDYF) that were infected with HLB. RESULTS: the HLB bacterial population followed the order WGF > TYBGF > WLYF > WDYF. And there were significant differences between each group of samples. Regarding the accumulation of chlorophyll and carotenoid, the chlorophyll-a content in WGF was the highest and in WDYF was the lowest. The content of chlorophyll-b in WGF was significantly higher than that in other three pericarps. There were significant differences in the total content of carotenoid between each group. WGF and TYBGF pericarps were low in phytoene, γ-carotene, ß-cryptoxanthin and apocarotenal, while other kinds of carotenoids were significantly higher than those in WDYF. And WLYF was only short of apocarotenal. We comprehensively compared the transcriptome and metabolite profiles of abnormal (WGF, TYBGF and WLYF) and normal (WDYF, control) pericarps. In total, 2,880, 2,782 and 1,053 differentially expressed genes (DEGs), including 121, 117 and 43 transcription factors were identified in the three comparisons, respectively. The qRT-PCR confirmed the expression levels of genes selected from transcriptome. Additionally, a total of 77 flavonoids and other phenylpropanoid-derived metabolites were identified in the three comparisons. Most (76.65 %) showed markedly lower abundances in the three comparisons. The phenylpropanoid biosynthesis pathway was the major enrichment pathway in the integrative analysis of metabolome and transcriptome profiles. CONCLUSIONS: Synthesizing the above analytical results, this study indicated that different color pericarps were associated with the reduced levels of some carotenoids and phenylpropanoids derivatives products and the down-regulation of proteins in flavonoids, phenylpropanoids derivatives biosynthesis pathway and the photosynthesis-antenna proteins.

Metabolomics Analysis of the Peels of Different Colored Citrus Fruits (Citrus reticulata cv. 'Shatangju') During the Maturation Period Based on UHPLC-QQQ-MS.

Citrus is a globally consumed fruit with great popularity. Mandarin (Citrus reticulata cv. 'Shatangju') is a local variety, and its planting area and yield are the greatest regarding fruit tree planting in Guangdong Province, China. However, its resistance to Huanglongbing (HLB) is weak. After infection by HLB, the fruits cannot develop normally. In this study, four kinds of fruits were classified as HBG, XQG, ZQG, and DHG, according to the color of their peels. The metabolomes of the three abnormally colored groups (HBG, XQG, and ZQG) and the normally colored group (DHG) were compared using a UPLC-QQQ-MS-based metabolomics approach. In total, 913 metabolites were identified and classified into 23 different categories, including phenylpropanoids and flavonoids; among them, 215 (HBG, 177; XQG, 124; and ZQG, 62) metabolites showed differential accumulation in the three comparison groups (HBG/XQG/ZQG versus DHG). A total of 2 unique metabolites, O-caffeoyl maltotriose and myricetin were detected only in DHG samples. When comparing HBG with DHG, there were 109 decreased and 68 increased metabolites; comparing XQG with DHG, there were 88 decreased and 36 increased metabolites; comparing ZQG with DHG, 41 metabolites were decreased, and 21 metabolites were increased. Metabolic pathway enrichment analysis of these differential metabolites showed significant enrichment of the "phenylpropanoid biosynthesis" pathway in all comparison groups. The hierarchical cluster analysis of the differential metabolites of the four groups showed a clear grouping patterns. The relative contents of three phenylpropanoids, four flavonoids, two alkaloids, one anthocyanin, and two other metabolites were significantly different between each comparison group. This study might provide fundamental insight for the isolation and identification of functional compounds from the peels of citrus fruit infected with HLB and for in-depth research on the effect of HLB on the formation of fruits pigment and the development of HLB-resistant citrus varieties.

Activation of RAW264.7 macrophages by an acidic polysaccharide derived from Citrus grandis 'Tomentosa'.

Citrus grandis 'Tomentosa' which is a special Citrus cultivar, has been employed as cough suppressant and expectorant in traditional Chinese medicine for thousands of years. The aim of this study is to investigate the immunomodulatory role of an acidic polysaccharide (designated as CGTP-AP) purified from C. grandis 'Tomentosa'. CGTP-AP showed effective immune activation in RAW264.7 macrophages at the concentration of 1-100 µg/mL. CGTP-AP could promote the release of NO in dose- and time-dependent manners. Enzyme-Linked Immunosorbent Assay (ELISA) and RT-PCR analysis demonstrated that CGTP-AP could stimulate the secretion of TNF-α and IL-6 in a dosage-dependent way. Western blot analysis and RT-PCR analysis indicated that CGTP-AP treatment could induce the iNOS and COX-2 expression in RAW264.7 macrophages. By conducting the inhibitors experiments, the activation of NF-κB and MAPK signaling pathways by CGTP-AP treatment was confirmed. Therefore, the present results declared that CGTP-AP could be a promising candidate as a potent immunomodulator for the application in future pharmaceutical development.

Involvement of LaeA in the regulation of conidia production and stress responses in Penicillium digitatum.

Involvement of LaeA in various biological processes of filamentous fungi has been demonstrated. However, its role in Penicillium digitatum, the causal agent of citrus postharvest green mold, remains unclear. In this study, a ΔPdLaeA mutant was constructed using homologous recombination. The production of conidia by the ΔPdLaeA mutant was reduced by half compared with that of the wild-type strain. The sensitivity of the ΔPdLaeA mutant increased under alkaline conditions. The virulence assay revealed that PdLaeA was dispensable for the virulence of P. digitatum. Comparative transcriptome analysis revealed that the function loss of PdLaeA resulted in the reduced expression of several secondary metabolite gene clusters. In addition, expression of several key regulators of conidiation (BrlA, FlbA, FlbC, FlbD, and FluG) was also downregulated in the ΔPdLaeA mutant. In summary, the present work demonstrated that PdLaeA was involved in the regulation of SM biosynthesis, as well as the development and environmental adaptation of P. digitatum.

Structural elucidation of an acidic polysaccharide from Citrus grandis 'Tomentosa' and its anti-proliferative effects on LOVO and SW620 cells.

Citrus grandis 'Tomentosa' (CGT) which is a region-famous medicinal and edible plant contains plentiful bioactive polysaccharides, however, its chemical structures and specific bioactivities still need to be further explored. In the present study, an acidic polysaccharide (CGTP-AP) was extracted and purified from the pulps of CGT. The structure elucidation and anti-cancer activity of CGTP-AP were investigated. Structure characterization indicated that CGTP-AP was a homogeneous heteropolysaccharide composed of arabinose, galactose and galacturonic acid in a molar ratio of 2.45:1:2.77, with an average molecular weight of 2721.68 kDa. Partial acid hydrolysis, methylation and NMR spectrometry revealed that the backbone of CGTP-AP mainly composed of (1 → 4)-α-D-galacturonan, while the branch principally consisted of (1 → 5)-α-L-Araf. In addition, CGTP-AP exhibited effective anti-proliferation against colon cancer cells LOVO and SW620 cells in dose- and time-dependent manners, with IC50 values of 5.55, 4.35 and 3.52 mg mL-1 after 24, 48 and 72 h, and 5.33, 3.63 and 2.97 mg mL-1 after 24, 48 and 72 h, respectively. This study indicated that CGTP-AP might be utilized as a promising food supplement for the patients of colon disorders.

Adenylyl cyclase is required for cAMP production, growth, conidial germination, and virulence in the citrus green mold pathogen Penicillium digitatum.

Penicillium digitatum is the causative agent of green mold decay on citrus fruit. The cAMP-mediated signaling pathway plays an important role in the transduction of extracellular signals and has been shown to regulate a wide range of developmental processes and pathogenicity in fungal pathogens. We cloned and characterized a Pdac1 gene of P. digitatum, which encodes a polypeptide similar to fungal adenylyl cyclases. Using a loss-of-function mutation in the Pdac1 gene we demonstrated a critical requirement for hyphal growth and conidial germination. Deletion of Pdac1 resulted in decreased accumulation of cAMP and down-regulation of genes encoding a G protein α subunit, both catalytic and regulatory subunits of PKA, and two transcriptional regulators StuA and Som1. Fungal mutants lacking Pdac1 produced abundant conidia, which failed to germinate effectively and displayed an elevated sensitivity to heat treatment. Pdac1 mutant failed to utilize carbohydrates effectively and thus displayed severe growth retardation on rich and synthetic media. Slow growth seen in the Pdac1 mutants could be due to a defect in nutrient sensing and acquisition. Quantitative RT-PCR analysis revealed that Pdac1 was primarily expressed at the early stage of infection. Fungal pathogenicity assayed on citrus fruit revealed that P. digitatum strains impaired for Pdac1 delayed lesion formation. Our results highlight important regulatory roles of adenylyl cyclase-mediated cAMP production in P. digitatum and provide insights into the critical role of cAMP in fungal growth, development and virulence.

Deletion of PdMit1, a homolog of yeast Csg1, affects growth and Ca(2+) sensitivity of the fungus Penicillium digitatum, but does not alter virulence.

GDP-mannose:inositol-phosphorylceramide (MIPC) and its derivatives are important for Ca(2+) sensitization of Saccharomyces cerevisiae and for the virulence of Candida albicans, but its role in the virulence of plant fungal pathogens remains unclear. In this study, we report the identification and functional characterization of PdMit1, the gene encoding MIPC synthase in Penicillium digitatum, one of the most important pathogens of postharvest citrus fruits. To understand the function of PdMit1, a PdMit1 deletion mutant was generated. Compared to its wild-type control, the PdMit1 deletion mutant exhibited slow radial growth, decreased conidia production and delayed conidial germination, suggesting that PdMit1 is important for the growth of mycelium, sporulation and conidial germination. The PdMit1 deletion mutant also showed hypersensitivity to Ca(2+). Treatment with 250 mmol/l Ca(2+) induced vacuole fusion in the wild-type strain, but not in the PdMit1 deletion mutant. Treatment with 250mmol/lCaCl2 upregulated three Ca(2+)-ATPase genes in the wild-type strain, and this was significantly inhibited in the PdMit1 deletion mutant. These results suggest that PdMit1 may have a role in regulating vacuole fusion and expression of Ca(2+)-ATPase genes by controlling biosynthesis of MIPC, and thereby imparts P. digitatum Ca(2+) tolerance. However, we found that PdMit1 is dispensable for virulence of P. digitatum.

A genomics based discovery of secondary metabolite biosynthetic gene clusters in Aspergillus ustus.

Secondary metabolites (SMs) produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic.

PdbrlA, PdabaA and PdwetA control distinct stages of conidiogenesis in Penicillium digitatum.

Penicillium digitatum is one of the most important citrus postharvest pathogens worldwide. Reproduction of massive asexual spores is the primary factor contributing to the epidemic of citrus green mold. To understand the molecular mechanisms underlying conidiogenesis in P. digitatum, we functionally characterized the Aspergillus nidulans orthologs of brlA, abaA and wetA. We showed that deletion of PdbrlA completely blocked formation of conidiophores, whereas deletion of PdabaA led to the formation of aberrant and non-functional phialides. The PdwetA mutant showed various defective phenotypes, such as abnormal conidia with loose cell walls, delayed germination and reduced tolerance to osmotic, detergent, heat shock and menadione stresses, but elevated resistance to H2O2. PdbrlA-influenced genes were identified by comparing global gene expression profiles between the wild-type and the PdbrlA deletion mutant during conidiation. Gene ontology analysis of these differentially expressed genes (DEGs) revealed the diverse roles of PdbrlA in metabolism, transportation and cell structure. Moreover, out of 39 genes previously reported to be involved in conidiogenesis in Aspergillus, mRNA levels of 14 genes were changed in ΔPdbrlA. Our results confirm the roles of brlA, abaA and wetA in P. digitatum conidiogenesis and provide new insights into the genetics of conidiation in filamentous fungi.

Glucosylceramides are required for mycelial growth and full virulence in Penicillium digitatum.

Glucosylceramides (GlcCers) are important lipid components of the membrane systems of eukaryotes. Recent studies have suggested the roles for GlcCers in regulating fungal growth and pathogenesis. In this study, we report the identification and functional characterization of PdGcs1, a gene encoding GlcCer synthase (GCS) essential for the biosynthesis of GlcCers, in Penicilliumdigitatum genome. We demonstrated that the deletion of PdGcs1 in P. digitatum resulted in the complete loss of production of GlcCer (d18:1/18:0 h) and GlcCer (d18:2/18:0 h), a decrease in vegetation growth and sporulation, and a delay in spore germination. The virulence of the PdGcs1 deletion mutant on citrus fruits was also impaired, as evidenced by the delayed occurrence of water soaking lesion and the formation of smaller size of lesion. These results suggest that PdGcs1 is a bona fide GCS that plays an important role in regulating cell growth, differentiation, and virulence of P. digitatum by controlling the biosynthesis of GlcCers.

Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum.

Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation (ATMT), but the frequencies are often very low. In the present study, we replaced the Ku80 homolog (a gene of the non-homologous end-joining (NHEJ) pathway) with the hygromycin resistance cassette (hph) by ATMT. No significant change in vegetative growth, conidiation, or pathogenicity was observed in Ku80-deficient strain (ΔPdKu80) of P. digitatum. However, using ΔPdKu80 as a targeting strain, the gene-targeting frequencies for both genes PdbrlA and PdmpkA were significantly increased. These results suggest that Ku80 plays an important role in homologous integration and the created ΔPdKu80 strain would be a good candidate for rapid gene function analysis in P. digitatum.

Os2 MAP kinase-mediated osmostress tolerance in Penicillium digitatum is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis.

High osmolarity glycerol (HOG) pathway is ubiquitously distributed among eukaryotic organisms and plays an important role in adaptation to changes in the environment. In this study, the Hog1 ortholog in Penicillium digitatum, designated Pdos2, was identified and characterized using a gene knock-out strategy. The ΔPdos2 mutant showed a considerably increased sensitivity to salt stress and cell wall-disturbing agents and a slightly increased resistance to fungicides iprodione and fludioxonil, indicating that Pdos2 is involved in response to hyperosmotic stress, regulation of cell wall integrity and sensitivity to fungicides iprodione and fludioxonil. Surprisingly, the mutant was not affected in response to oxidative stress caused by H2O2. The average lesion size in citrus fruits caused by ΔPdos2 mutant was smaller (approximately 25.0% reduction) than that caused by the wild-type strain of P. digitatum at 4 days post inoculation, which suggests that Pdos2 is needed for full virulence of P. digitatum. Interestingly, in the presence of 0.7 M NaCl, the glycerol content was remarkably increased and the ergosterol was decreased in mycelia of the wide-type P. digitatum, whereas the glycerol content was only slightly increased and the ergosterol content remained stable in the ΔPdos2 mutant, suggesting that Pdos2-mediated osmotic adaption is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis.

Genomewide investigation into DNA elements and ABC transporters involved in imazalil resistance in Penicillium digitatum.

Penicillium digitatum, causing citrus green mold, is one of the most devastating pathogenic fungi for postharvest fruits. The disease control is becoming less efficient because of the dispersal of fungicide-resistant strains. However, genome-scale analyses of its resistance mechanism are scarce. In this work, we sequenced the whole genome of the R1 genotype strain Pd01-ZJU and investigated the genes and DNA elements highly associated with drug resistance. Variation in DNA elements related to drug resistance between P. digitatum strains was revealed in both copy number and chromosomal location, indicating that their recent and frequent translocation might have contributed to environmental adaptation. In addition, ABC transporter proteins in Pd01-ZJU were characterized, and the roles of typical subfamilies (ABCG, ABCC, and ABCB) in imazalil resistance were explored using real-time PCR. Seven ABC proteins, including the previously characterized PMR1 and PMR5, were induced by imazalil, which suggests a role in drug resistance. In summary, this work presents genome information of the R1 genotype P. digitatum and systematically investigates DNA elements and ABC proteins associated with imazalil resistance for the first time, which would be indicative for studying resistant mechanisms in other pathogenic fungi.