Identification and characterization of Cercospora beticola necrosis-inducing effector CbNip1.

Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin-initiated formation of reactive oxygen species is light-dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light-dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1-triggered necrosis, as light exposure delayed CbNip1-triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola-sugar beet disease process.

Inhibitors of tri- and tetra- polyamine oxidation, but not diamine oxidation, impair the initial stages of wound-induced suberization.

The mechanisms regulating, and modulating potato wound-healing processes are of great importance in reducing tuber infections, reducing shrinkage and maintaining quality and nutritional value for growers and consumers. Wound-induced changes in tuber polyamine metabolism have been linked to the modulation of wound healing (WH) and in possibly providing the crucial amount of H2O2 required for suberization processes. In this investigation we determined the effect of inhibition of specific steps within the pathway of polyamine metabolism on polyamine content and the initial accumulation of suberin polyphenolics (SPP) during WH. The accumulation of SPP represents a critical part of the beginning or inchoate phase of tuber WH during closing-layer formation because it serves as a barrier to bacterial infection and is a requisite for the accumulation of suberin polyaliphatics which provide the barrier to fungal infection. Results showed that the inhibitor treatments that caused changes in polyamine content generally did not influence wound-induced accumulation of SPP. Such lack of correlation was found for inhibitors involved in metabolism and oxidation of putrescine (arginine decarboxylase, ornithine decarboxylase, and diamine oxidase). However, accumulation of SPP was dramatically reduced by treatment with guazatine, a potent inhibitor of polyamine oxidase (PAO), and methylglyoxal-bis(guanylhydrazone), a putative inhibitor of S-adenosylmethione decarboxylase which may also cross-react to inhibit PAO. The mode of action of these inhibitors is presumed to be blockage of essential H2O2 production within the WH cell wall. These results are of great importance in understanding the mechanisms modulating WH and ultimately controlling related infections and associated postharvest losses.

Prevalence and Distribution of Beet Necrotic Yellow Vein Virus Strains in North Dakota and Minnesota.

Beet necrotic yellow vein virus (BNYVV) is the causal agent of rhizomania, a disease of global importance to the sugar beet industry. The most widely implemented resistance gene to rhizomania to date is Rz1, but resistance has been circumvented by resistance-breaking (RB) isolates worldwide. In an effort to gain greater understanding of the distribution of BNYVV and the nature of RB isolates in Minnesota and eastern North Dakota, sugar beet plants were grown in 594 soil samples obtained from production fields and subsequently were analyzed for the presence of BNYVV as well as coding variability in the viral P25 gene, the gene previously implicated in the RB pathotype. Baiting of virus from the soil with sugar beet varieties possessing no known resistance to rhizomania resulted in a disease incidence level of 10.6% in the region examined. Parallel baiting analysis of sugar beet genotypes possessing Rz1, the more recently introgressed Rz2, and with the combination of Rz1 + Rz2 resulted in a disease incidence level of 4.2, 1.0, and 0.8%, respectively. Virus sequences recovered from sugar beet bait plants possessing resistance genes Rz1 and/or Rz2 exhibited reduced genetic diversity in the P25 gene relative to those recovered from the susceptible genotype while confirming the hypervariable nature of the coding for amino acids (AAs) at position 67 and 68 in the P25 protein. In contrast to previous reports, we did not find an association between any one specific AA signature at these positions and the ability to circumvent Rz1-mediated resistance. The data document ongoing virulence development in BNYVV populations to previously resistant varieties and provide a baseline for the analysis of genetic change in the virus population that may accompany the implementation of new resistance genes to manage rhizomania.

Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum.

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.

Wounding induces changes in cytokinin and auxin content in potato tuber, but does not induce formation of gibberellins.

Cytokinin, auxin and gibberellin contents in resting and wound-responding potato tubers have not been fully determined and coordinated with wound-healing processes. Using a well-defined wound-healing model system, hormone content and expression of genes associated with hormone turnover were determined in tubers following wounding. Changes in hormone content were coordinated with: (I) formation and completion of the wound closing layer (0-5/6 days), and (II) initiation of phellogen and wound periderm formation (∼ 7 days). Quantifiable amounts of biologically active cytokinins (Z, DZ and IP) were not detected in resting or wound-responding tubers. However, the precursor IPA and catabolic product c-ZOG were found in small amounts in resting and wound-responding tubers. Wound-induced activation of cytokinin biosynthesis was suggested by an increase in t-ZR and c-ZR content at 0.5 days and large increases in IPA and c-ZR content by 3 days and throughout 7 days after wounding suggesting roles in II, but little or no role in I. Expression of key genes involved in cytokinin metabolism followed similar profiles with transcripts decreasing through 3 days and then increasing at 5-7 days after wounding. Both free IAA and IAA-Asp were present in resting tubers. While IAA-Asp was no longer present by 3 days after wounding, IAA content nearly doubled by 5 days and was more than 4-fold greater at 7 days compared to that in resting tuber (0 day) suggesting roles in II, but little or no role in I. Gibberellins were not present in quantifiable amounts in resting or wound-responding tubers. These results suggest that bio-active cytokinins are wound-induced, but their residency is temporal and highly regulated. The transient presence of active cytokinins and corresponding increases in IAA content strongly suggest their involvement in the regulation of wound periderm development. The absence of gibberellins indicates that they are not a regulatory component of wound-healing processes.

Wounding induces changes in tuber polyamine content, polyamine metabolic gene expression, and enzyme activity during closing layer formation and initiation of wound periderm formation.

Tuber wound-healing processes are complex, and the associated regulation and modulation of these processes are poorly understood. Polyamines (PA) are involved in modulating a variety of responses to biotic and abiotic plant stresses and have been suggested to be involved in tuber wound responses. However, the time course of wound-induced changes in tuber PA content, activity of key biosynthetic enzymes and associated gene expression has not been determined and coordinated with major wound-healing processes. The objective of this study was to determine these wound-induced changes and their coordination with wound-healing processes. Wounding induced increases in putrescine (Put) and spermidine (Spd), but had only minor effects on spermine (Spm) content during the 168 h time course which encompassed the initiation and completion of the closing layer formation, and the initiation of cell division and wound periderm formation. As determinants of the first committed step in PA biosynthesis, arginine and ornithine decarboxylase (ADC and ODC, respectively) activities were below levels of detectability in resting tubers and expression of genes encoding these two enzymes was low. Within 6h of wounding, increases in the in vitro activities of ADC and ODC and expression of their cognate genes were observed. Expression of a gene encoding S-adenosylmethionine decarboxylase, required for Spd and Spm biosynthesis, was also increased 6h after wounding and remained elevated throughout the time course. Expression of a polyamine catabolic gene, encoding polyamine oxidase, was down-regulated after wounding. Results indicated a rapid wound-induced increase in PA biosynthesis during closing layer formation and the time of nuclei entry and exit from S-phase. PA content remained elevated as wound-induced cells became meristematic and initiated formation of the wound periderm suggesting sustained involvement in wound-healing.

Kinetics and localization of wound-induced DNA biosynthesis in potato tuber.

Tuber wounding induces a cascade of biological responses that are involved in processes required to heal and protect surviving plant tissues. Little is known about the coordination of these processes, including essential wound-induced DNA synthesis, yet they play critical roles in maintaining marketability of the harvested crop and tubers cut for seed. A sensitive "Click-iT EdU Assay" employing incorporation of the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), in conjunction with 4',6-diamindino-2-phenylindole (DAPI) counter labeling, was employed to objectively identify and determine the time course and spatial distribution of tuber nuclei that were wound-induced to enter S-phase of the cell cycle. Both labeling procedures are rapid and sensitive in situ. Following wounding, EdU incorporation (indicating DNA synthesis) was not detectable until after 12h, rapidly reached a maximum at about 18h and then declined to near zero at 48h. About 28% of the nuclei were EdU labeled at 18h reflecting the proportion of cells in S-phase of the cell cycle. During the ∼30h in which induced cells were progressing through S-phase, de novo DNA synthesis extended 7-8 cell layers below the wound surface. Cessation of nuclear DNA synthesis occurred about 4 d prior to completion of wound closing layer formation. Initiation of wound periderm development followed at 7 d, i.e. about 5 d after cessation of nuclear DNA biosynthesis; at this time the phellogen developed and meristematic activity was detected via the production of new phellem cells. Collectively, these results provide new insight into the coordination of wound-induced nucleic acid synthesis with associated tuber wound-healing processes.

Wounding of potato tubers induces increases in ABA biosynthesis and catabolism and alters expression of ABA metabolic genes.

The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato tubers. An increase in ABA and ABA metabolite content was observed 48h after wounding and remained elevated through 96h. Wounding induced dramatic increases in the expression of the ABA metabolic genes encoding zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and ABA-8'-hydroxylase. Although the patterns of wound-induced expression of individual genes varied, increased gene expression was observed within 3h of wounding and remained elevated through 96h. An apparent correlation between expression of the gene encoding ZEP and the increase in ABA content suggested that the wound-induced increase in ABA biosynthesis was regulated by both substrate availability and increased NCED activity. Suppression of wound-induced jasmonic acid accumulation by rinsing the wounded tissue with water did not inhibit the subsequent increase in ABA content. Exogenous ethylene completely suppressed the wound-induced increase in ABA content and dramatically reduced wound-induced up-regulation of ABA metabolic genes. This study is the first to identify the molecular bases for increased ABA accumulation following physical trauma in potato tubers and highlights the complex physiological interactions between various wound-induced hormones.

Molecular and cytological aspects of native periderm maturation in potato tubers.

Mature native periderm that exhibits resistance to excoriation (RE) is the primary defense for potato tubers against abiotic and biotic challenges. However, little is known about the physiology of periderm maturation and associated gene expressions. In this study, periderm maturation events and associated gene expressions were determined in tubers of two diverse potato genotypes (NDTX4271-5R (ND) and Russet Burbank (RB); 2008 and 2009 crops) at four harvest maturities ranging from immature (non-senesced vines and low RE) to mature (senesced vines and high RE). Approximately 104 d after planting, the fine balance of accumulation and loss of periderm phellem cell layers showed signs of subsiding, indicating cessation of cell division by the phellogen. Phellogen radial cell walls thickened as periderm matured throughout the harvests, increasing RE/skin-set. In both genotypes, the cell cycle gene cyclin-dependent kinase B (StCDKB) rapidly down-regulated after the second harvest coinciding with apparent cessation of cell division. Expression patterns of genes encoding epidermal growth factor binding protein (StEBP) and cyclin-dependent kinase regulatory subunit (StCKS1At) were less indicative of phellogen inactivation and periderm maturation. Genes encoding the structural cell wall proteins extensin (StExt1) for ND and extensin-like (StExtlk) for ND and RB remained up-regulated respectively by the second harvest, suggesting involvement with completion of phellem cell accumulation and on-set of periderm maturation. The expression of genes encoding pectin methyl esterase (StPME), StExt1 and a cell wall strengthening "tyrosine-and lysine-rich protein" (StTLRP) increased in phellogen cells from later harvests of ND tubers, but were down regulated in RB tubers; this suggests roles in phellem cell generation and completion of delayed cell wall development in non-meristematic phellogen cells of ND, a red skinned phenotype. StCDKB and StPrePME genes were rapidly down-regulated by the third harvest for both genotypes. Collectively, these results suggest that down-regulation of these genes coordinates with on-set of periderm maturation and skin-set progression.

Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall thickening after cessation of phellogen cell division.