Pathogenicity and diversity of vegetative compatibility of Fusarium verticillioides

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Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci., Matica Srpska Novi Sad, ¥ 113, 103—111, 2007 UDC 632.4:633.11 632.4:633.15 V e s n a S. K r n j a j a1 , J e l e n a T. L e v i ã2 , S l a v i c a Ÿ. S t a n k o v i ã2 , Z o r i c a M. T o m i ã1 1 Institute for Animal Husbandry, Autoput 16, 11081 Belgrade, Republic of Serbia 2 Maize Research Institute, Zemun Polje, Slobodana Bajiãa 1, 11185 Belgrade, Republic of Serbia PATHOGENICITY AND DIVERSITY OF VEGETATIVE COMPATIBILITY OF FUSARIUM VERTICILLIOIDES ABSTRACT: Pathogenicity of 10 Fusarium verticillioides isolates, originated from grain of wheat (five isolates) and maize (five isolates), were studied under greenhouse conditions. Based on different parameters of the pathogenicity estimate (a scale for % of nonemerged plants, % of survived plants, plant vigour — the growth and dry weight of roots and epicotyls and disease severity) it was determined that all F. verticillioides isolates expressed a different degree of pathogenicity. According to % of nonemerged plants six, three and one F. verticillioides isolates expressed low, moderate and high deegre of pathogenicity, respectively. All F. verticillioides isolates reduced the plant survival rate and vigour, while the disease severity ranged from 2.0 to 3.54. Two types of nit mutants, nit1 and NitM, were obtained by the use of the method of vegetative compatibility. The frequency of nit1 mutants was greater (58.79%) than the frequency of NitM mutants (5.77%). A total of 10 vegetative compatibility groups (VCGs) of F. verticillioides were established in the complementation tests. These results point out to a high genetic diversity of F. verticillioides population. KEY WORDS: Fusarium verticillioides, pathogenicity, vegetative compatibility INTRODUCTION Fusarium verticillioides (Sacc.) Nirenberg (syn. Fusarium moniliforme Scheldon) is a widely distributed pathogen of maize (Zea mays L.) and many other plant species. In Serbia, F. verticillioides, as a pathogen of grain, was identified on maize, wheat and sorghum up to 77.8% (L e v i ã et al., 1997, 2003), 10% (D o p u ð a and L e v i ã, 2004) and 7.5% (L e v i ã et al., 2006), respectively. The epidemiological studies show that F. verticillioides has one comparative advantage over other species of the genus Fusarium, especially in relation to F. graminearum Schwabe, as it requires a greater range of temperatures ( R e i d et al., 1999) and humidity (S c h n e i d e r and P e n d e r y 1983) for 103 its development, hence the competitiveness of the fungus will not change in different environments. It is typical of this species to colonise the plant tissue and to remain at the dormant stage or at the endophytic stage, as long as the tissue is healthy and active (M u n k v o l d and D e s j a r d i n s, 1997). It is difficult to discuss with certainty the role of F. verticillioides in the etiology of seedling diseases, as seedling infections and the disease development are produced by the seed affected by a disease or a contaminated soil, and depend on the temperature during the maize growing period (K o m m e d a h l and W i n d e l s, 1981). F. verticillioides does not affect seed germination at the endophytic stage, but it affects the thickness, height, weight and leaf length of seedlings developed from infected seeds (Y a t e s et al., 1997). On the other hand, some strains of this fungus can even stimulate an earlier growth of seedlings. L e v i ã (2000) established that the frequency of occurrence of Fusarium species is not always correlated with their effects on seed germination. According to this author, the following species are most often isolated from maize grain: F. verticillioides (50.2%), F. subglutinans (Wollenw. & Reinking) Nelson (45.6%) and then F. proliferatum (Matsushima) Nirenberg (7.9%). However, germination of seeds infected with F. subglutinans, F. proliferatum and F. verticillioides amounted to 15.3%, 23.4% and 32.6%, respectively. The characterization of F. verticillioides isolates can be done on the basis of the seedling pathogenicity test or vegetative compatibility, since it was determined that isolates of the similar pathogenicity belonged to the same vegetative compatibility group (VCG) (K l e i n and C o r r e l l, 2001). Therefore, if a rapid method of the VCG identification is developed and rapid analyses of the population strain evaluation are provided, then the VCG pathogen strain identification can replace the pathogenicity test, which is time — consuming and requires specific, control led conditions, depending on a plant species. Considering the economic importance of F. verticillioides, pathogenicity of F. verticillioides isolates, originating in maize and wheat grown at different locations in Serbia, to maize seedlings and their vegetative compatibility were observed in this study. MATERIAL AND METHODS Fungal isolates Ten isolates of F. verticillioides were used to perform pathogenicity and vegetative compatibility tests. Five isolates originated from grain of commercial maize hybrids grown in the vicinity of Belgrade—Zemun, and five isolates originated from grain of wheat varieties grown at different locations in Serbia (Table 1). Isolates were identified as F. verticillioides using of the procedure outlined by N e l s o n et al. (1983) and B u r g e s s et al. (1994). 104 Tab. 1 — F. verticilioides isolates tested for pathogenicity to maize seedlings under greenhouse conditions and vegetative compatibility No. Isolate 1. MGA-7 2. MGD-4 3. MGE-5 4. MGG-13 5. MGI-1 6. MRIZP-201 7. MRIZP-237 8. MRIZP-570 9. MRIZP-748 10. MRIZP-830 Origin Belgrade—Zemun Belgrade—Zemun Belgrade—Zemun Belgrade—Zemun Belgrade—Zemun Inðija Inðija Ruma Loznica Sombor Host Commercial maize hybrid Commercial maize hybrid Commercial maize hybrid Commercial maize hybrid Commercial maize hybrid Evropa 90 (wheat variety) Pobeda (wheat variety) Renesansa (wheat variety) Simonida (wheat variety) Evropa 90 (wheat variety) Selected cultures were initiated from single conidia and stored on PDA slants at 4°C, until use for the pathogenicity test and vegetative compatibility. Pathogenicity test with maize seedlings An insignificantly modified method described by M o l o t and S i m o n e (1967) was followed for estimations of pathogenicity of F. verticillioides isolates. Petri dishes with the two-layer filter paper, instead of flasks, and sterile quartz sand, instead of soil, were used for the development of the fungus and artificial inoculation of seeds. A total of 45 maize seeds, surface-sterilised with sodium hypochlorite per isolate were inoculated in the sterile Petri dishes (ø 100 mm) with 30 ml of spore suspension (2—3 x 106 spore ml—1). The spore suspension was prepared from 7—10 old isolates cultured on the PDA at room temperature. Inoculated and non-inoculated (control) maize seeds were incubated at 22°C for two days and at 10°C for three days, and then planted into flats (40 x 18 x 16 cm) with sterile quartz sand, watered and incubated at 24—26°C. Maize seeds were inoculated for two weeks and the following was determined: degree of pathogenicity, length (cm) and dry weight (g) of seedling roots and epicotyls. In this study, the degree of pathogenicity was defined on the basis of nonemerged plants (%), which was an outcome of seeds that had never germinated, and germinated seeds with completely rotted shoots. According to this parameter, the isolates were classified into five categories based on the scale described by M a ç k a (1989) (Table 2). Tab. 2 — The scale for the estimation of pathogenicity of F. verticillioides isolates Percentage of nonemerged plants 0—10% 11—20% 21—40% 41—60% 61—80% 81—100% Degree of pathogenicity not pathogenic very low pathogenic low pathogenic moderate pathogenic high pathogenic very high pathogenic 105 Disease severity was also used as a measurement of pathogenicity of isolates, and was rated by a six-class scale, in which 0 = healthy root and epicotyl, and 5 = nongerminated seed, or completely rotted root and shoot. The length of each seedling from the seed attachment site to the top of the longest root and leaves was measured (cm). The detached root and epicotyl per replicate were dried at 60°C for 24 hours and then, their weights (g) were measured. Means were compared by Duncan's multiple range test. Vegetative compatibility groups Methods described by C o r r e l l et al. (1987) and K e d e r a et al. (1994) were used to isolate and characterize nit-mutants and their mutual pairing in order to determine vegetative compatibility of the studied F. verticillioides isolates. The excised pieces of mycelia were planted on the minimum me- dium (a basal medium amended with 30 g KClO3, 2 g NaNO3, and 1.6 g L-asparagine) for the selection of mutants (sectors). The basal medium conta- ins 1.0 g KH2PO4; 0.5 g MgSO4 x 7H2O; 0.5 g KCl; 10 mg FeSO4 x 7H2O; 0.2 ml sterile solution of microelemenats; 30.0 g of sucrose; 20.0 g of Difco agar; 1000 ml of distilled water. Pieces of hyphae and loose growing sectors were transferred to the basal NaNO2 and hypoxantine) in medium order to with different determine the tnyiptreogoefnthsoeunrcite-sm(uNtaanNt Oo3n, the basis of a phenotype (P u h a l l a, 1985). Complementary nit1 and NitM mutants from each of 10 F. vertillicioides isolates were paired on the minimum medium (MM) in all possible combinati- ons to perform complementation tests among the isolates. The nit mutants grew very sparsely across the medium, but complementation of auxotrophic mutants was indicated by a line of a vigorous growth where the mutants inter- acted. RESULTS AND DISCUSSION Pathogenicity All observed F. verticillioides isolates affected the survival rate and vigour of plants. Out of 10 F. verticillioides isolates tested under greenhouse conditions six, three and one isolates were low (26.67—40.0% of nonemerged plants), moderate (48.87—55.53% of nonemerged plants) and high (62.20% of nonemerged plants) pathogenic (Table 3). The isolate MGG-13, originated from maize grain, was estimated as high pathogenic as it reduced germination by 62.20%. The same isolate was significantly more pathogenic than the remaining isolates, as determined disease severity (3.69) was the highest and the survival rate (37.80%) and plant vigour were the lowest (Table 4). 106 Tab. 3 — F. verticillioides isolates classified on the basis of a percentage of nonemerged plants Isolate MGA-7 MGD-4 MGE-5 MGG-13 MGI-1 MRIZP-201 MRIZP-237 MRIZP-570 MRIZP-748 MRIZP-830 Control Nonemerged plants (%) 31.13 40.00 26.67 62.20 55.53 48.87 37.80 51.13 40.00 35.54 4.47 Degree of pathogenicity low pathogenic low pathogenic low pathogenic high pathogenic moderate pathogenic moderate pathogenic low pathogenic moderate pathogenic low pathogenic low pathogenic The survival rate of seedlings developed from inoculated seeds with different isolates of F. verticillioides varied from 37.80% (MGG-13) to 73.33% (MGE-5), which was significantly lower than in the control (95.53%) (Table 4). The observed isolates affected the reduction of the root growth in comparison with the epicotyl growth. The isolate MGI-1, originated from maize, as well as, isolates MRIZP-201 and MRIZP-570, originated from wheat, expressed similar pathogenicity, which was particularly established on the basis of the root growth. The isolate MGD-4 showed peculiar behaviour, as disease severity caused to seedlings was high (3.18), which made it similar to the high pathogenic isolate (MGG-13), but due to a relatively high survival rate of plants (60.00%), it was estimated as low pathogenic (Table 4). Tab. 4 — Effect of F. verticillioides isolates on maize seedlings growing from artificially infected seeds under greenhouse conditions No. Isolate 1. MGA-7 2. MGD-4 3. MGE-5 4. MGG-13 5. MGI-1 6. MRIZP-201 7. MRIZP-237 8. MRIZP-570 9. MRIZP-748 10. MRIZP-830 Average 11. Control LSD (0.05) LSD (0.01) Plant survival* (%) 68.87bc 60.00bcd 73.33ab 37.80d 44.47cd 51.13bcd 62.20bcd 48.87bcd 60.00bcd 64.46bcd 57.64 95.53a 3.574 4.876 Plant vigour* Length (cm) Dry weight (g) Root Epicotyl Root Epicotyl 18.73bcd 15.87cd 23.74b 11.42d 13.79d 11.26d 23.22bc 15.34d 17.77bcd 13.13d 14.71bc 12.14bc 16.86ab 8.98c 9.87c 12.15bc 18.26ab 13.52bc 13.41bc 13.18bc 2.10bcd 1.37cde 2.23bc 1.10e 1.33de 1.00e 2.00bcd 1.23de 2.63b 1.80bcde 0.800abc 0.500bc 0.767abc 0.400bc 0.367c 0.833ab 0.833ab 0.56bc 0.600bc 0.633abc 16.43 13.30 1.67 0.629 31.62a 22.49a 3.97a 1.067a 6.864 9.364 5.411 7.382 0.800 1.092 0.388 0.529 Disease severity* 2.66bcd 3.18abc 2.00d 3.69a 3.41abc 3.54ab 2.54bcd 3.02abc 2.40cd 2.48cd 2.85 0.10e 0.898 1.225 * Values of column followed by the same letter(s) are not significantly different (P = 0.05) according to Duncan's multiple range test. 107 Our results on pathogenicity of F. verticillioides are in accordance with the results obtained by D e s j a r d i n s et al. (1995) and M u n k v o l d and C a r l t o n (1997). Vegetative compatibility Mutants nit1 and NitM, with prevalence of nit1 (58.79%) over NitM (5.77%) (Table 5), were isolated from the observed isolates of F. verticillioides. According to the literature data (K l i t t i c h and L e s l i e, 1988), the frequency of mutants nit1 is higher than the frequency of other types of nit mutants. Tab. 5 — Frequency of nit1 and NitM mutants in the studied F. verticillioides isolates Isolate MGA-7 MGD-4 MGE-5 MGG-13 MGI-1 MRIZP-201 MRIZP-237 MRIZP-570 MRIZP-748 MRIZP-830 Average nit1 (%) 92.86 70.00 55.00 40.00 65.00 45.00 62.50 50.00 67.50 40.00 58.79 NitM (%) 4.29 15.00 5.00 5.00 5.00 6.67 1.25 7.14 5.00 3.33 5.77 Ten vegetative compatible groups (VCGs) of F. verticillioides were established on the basis of the complementation test among the isolates in all possible combinations. These results point out to a high genetic diversity of the population of this fungus pathogenic to maize. Similar results were stated by C h u l z e et al. (2000). CONCLUSION All studied F. verticillioides isolates originated from wheat (five isolates) and maize grain (five isolates) expressed pathogenicity to maize seedlings. According to the percentage of nonemerged plants, it was established that six, three and one isolates expressed low, moderate and high pathogenicity. The survival rate (%) and vigour (growth and dry weight of roots and epicotyls) of plants that were developed from inoculated seeds, were significantly reduced (approximately two times) in comparison with the control. There was a tendency for isolates from different hosts to have similar values for pathogenicity. These results are of a practical importance from the aspects of maize and wheat crop rotation and for the success of breeding for resistance to F. verticillioides. 108 The analysis of the results on pathogenicity obtained on the basis of the scale for the % nonemerged plants, plant survival rate (%), vigour (growth and dry weight of roots and epicotyls) and disease severity, shows a concurrence in defining moderate and high pathogenicity of isolates, while there was a certain nonconformance among these results in relation to low pathogenicity defining. Nevertheless, a selection of parameters, such as the scale for % of nonemerged plants, is a simple and good choice for the characterisation of pathogenicity degree of all F. verticillioides isolates. Two types of nit mutants, nit1 and NitM, were obtained by the use of the method of vegetative compatibility. The frequency of nit1 mutants was greater (58.79%) than the frequency of NitM mutants (5.77%). A total of 10 VCGs of F. verticillioides were determined in the complementation tests. This number of vegetative compatible groups indicates a high genetic diversity of the observed F. verticillioides population. ACKNOWLEDGEMENTS This paper is a part of the investigations implemented within the scope of the project No. TR-6826B financially supported by the Ministry of Science and Environmental Protection of the Republic of Serbia. REFERENCES B u r g e s s, L. W., S u m m e r e l l, B. A., B u l l o c k, S., G o t t, K. P., B a c k h o u s e, D. (1994): Laboratory for Fusarium Research, Third Edition, Fusarium Research Laboratory, Department of Crop Sciences, University of Sydney and Royal Botanic Garden, Sydney, 133. C h u l z e, S. N., R a m i r e z, M. L., T o r r e s, A., L e s l i e, J. F. (2000): Genetic variation in Fusarium Section Liseola from no-till maize in Argentina, Appl. Environ. Microbiol. 66 (12): 5312—5315. C o r r e l l, J. C., K l i t t i c h, C. J. R., L e s l i e, J. F. (1987): Nitrate nonutilizing mutants of Fusarium oxysporum and their use in vegetative compatibility tests, Phytopathology 77: 1640—1646. D e s j a r d i n s, A. E., P l a t t n e r, R. D., N e l s e n, T. C., L e s l i e, J. F. (1995): Genetic analysis of fumonisin production and virulence of Gibberella fujikuroi mating population A (Fusarium moniliforme) on maize (Zea mais) seedlings, Appl. Environ. Microbiol. 61 (1): 79—86. D o p u ð a, M., J. L e v i ã (2004): Sastav mikobiote (Fusaria) semena pšenice na podruåju Srema, Zb. radova str. 112, 5. Kong. z. bilja, Zlatibor, 22—26. novembar 2004. K e d e r a, C. J., L e s l i e, J. F., C l a f l i n, L. E. (1994): Genetic diversity of Fusarium section Liseola (Gibberella fujikuroi) in individual corn stalks, Phytopathology 84: 603—607. K l e i n, K. K., C o r r e l l, J. C. (2001): Vegetative compatibility group diversity in Fusarium, Chapter 6, 83—96, in: Summerell, B. A., Leslie, J. F., Backhouse, D., 109 Bryden, W. L., Burgess, L. W. (ed.), Fusarium — Paul E. Nelson Memorial Symposium, AS Press, The American Phytopathological Society, St. Paul, Minnesota, 392. equilibrium value of MeCpG steps (,+14 deg.) [31,44]. In comparison, methylation has a significantly lower stability cost when happening at major groove positions, such as 211 and 21 base pair from dyad (mutations 9 and 12), where the roll of the nucleosome bound conformation (+10 deg.) is more compatible with the equilibrium geometry of MeCpG steps. The nucleosome destabilizing effect of cytosine methylation increases with the number of methylated cytosines, following the same position dependence as the single methylations. The multiple-methylation case reveals that each major groove meth- PLOS Computational Biology | 3 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning ylation destabilizes the nucleosome by around 1 kJ/mol (close to the average estimate of 2 kJ/mol obtained for from individual methylation studies), while each minor groove methylation destabilizes it by up to 5 kJ/mol (average free energy as single mutation is around 6 kJ/mol). This energetic position-dependence is the reverse of what was observed in a recent FRET/SAXS study [30]. The differences can be attributed to the use of different ionic conditions and different sequences: a modified Widom-601 sequence of 157 bp, which already contains multiple CpG steps in mixed orientations, and which could assume different positioning due to the introduction of new CpG steps and by effect of the methylation. The analysis of our trajectories reveals a larger root mean square deviation (RMSD) and fluctuation (RMSF; see Figures S2– S3 in Text S1) for the methylated nucleosomes, but failed to detect any systematic change in DNA geometry or in intermolecular DNA-histone energy related to methylation (Fig. S1B, S1C, S4–S6 in Text S1). The hydrophobic effect should favor orientation of the methyl group out from the solvent but this effect alone is not likely to justify the positional dependent stability changes in Figure 2, as the differential solvation of the methyl groups in the bound and unbound states is only in the order of a fraction of a water molecule (Figure S5 in Text S1). We find however, a reasonable correlation between methylation-induced changes in hydrogen bond and stacking interactions of the bases and the change in nucleosome stability (see Figure S6 in Text S1). This finding suggests that methylation-induced nucleosome destabilization is related to the poorer ability of methylated DNA to fit into the required conformation for DNA in a nucleosome. Changes in the elastic deformation energy between methylated and un-methylated DNA correlate with nucleosomal differential binding free energies To further analyze the idea that methylation-induced nucleosome destabilization is connected to a worse fit of methylated DNA into the required nucleosome-bound conformation, we computed the elastic energy of the nucleosomal DNA using a harmonic deformation method [36,37,44]. This method provides a rough estimate of the energy required to deform a DNA fiber to adopt the super helical conformation in the nucleosome (full details in Suppl. Information Text S1). As shown in Figure 2, there is an evident correlation between the increase that methylation produces in the elastic deformation energy (DDE def.) and the free energy variation (DDG bind.) computed from MD/TI calculations. Clearly, methylation increases the stiffness of the CpG step [31], raising the energy cost required to wrap DNA around the histone octamers. This extra energy cost will be smaller in regions of high positive roll (naked DNA MeCpG steps have a higher roll than CpG steps [31]) than in regions of high negative roll. Thus, simple elastic considerations explain why methylation is better tolerated when the DNA faces the histones through the major groove (where positive roll is required) that when it faces histones through the minor groove (where negative roll is required). Nucleosome methylation can give rise to nucleosome repositioning We have established that methylation affects the wrapping of DNA in nucleosomes, but how does this translate into chromatin structure? As noted above, accumulation of minor groove methylations strongly destabilizes the nucleosome, and could trigger nucleosome unfolding, or notable changes in positioning or phasing of DNA around the histone core. While accumulation of methylations might be well tolerated if placed in favorable positions, accumulation in unfavorable positions would destabilize the nucleosome, which might trigger changes in chromatin structure. Chromatin could in fact react in two different ways in response to significant levels of methylation in unfavorable positions: i) the DNA could either detach from the histone core, leading to nucleosome eviction or nucleosome repositioning, or ii) the DNA could rotate around the histone core, changing its phase to place MeCpG steps in favorable positions. Both effects are anticipated to alter DNA accessibility and impact gene expression regulation. The sub-microsecond time scale of our MD trajectories of methylated DNAs bound to nucleosomes is not large enough to capture these effects, but clear trends are visible in cases of multiple mutations occurring in unfavorable positions, where unmethylated and methylated DNA sequences are out of phase by around 28 degrees (Figure S7 in Text S1). Due to this repositioning, large or small, DNA could move and the nucleosome structure could assume a more compact and distorted conformation, as detected by Lee and Lee [29], or a slightly open conformation as found in Jimenez-Useche et al. [30]. Using the harmonic deformation method, we additionally predicted the change in stability induced by cytosine methylation for millions of different nucleosomal DNA sequences. Consistently with our calculations, we used two extreme scenarios to prepare our DNA sequences (see Fig. 3): i) all positions where the minor grooves contact the histone core are occupied by CpG steps, and ii) all positions where the major grooves contact the histone core are occupied by CpG steps. We then computed the elastic energy required to wrap the DNA around the histone proteins in unmethylated and methylated states, and, as expected, observed that methylation disfavors DNA wrapping (Figure 3A). We have rescaled the elastic energy differences with a factor of 0.23 to match the DDG prediction in figure 2B. In agreement with the rest of our results, our analysis confirms that the effect of methylation is position-dependent. In fact, the overall difference between the two extreme methylation scenarios (all-in-minor vs all-in-major) is larger than 60 kJ/mol, the average difference being around 15 kJ/ mol. We have also computed the elastic energy differences for a million sequences with CpG/MeCpG steps positioned at all possible intermediate locations with respect to the position (figure 3B). The large differences between the extreme cases can induce rotations of DNA around the histone core, shifting its phase to allow the placement of the methylated CpG steps facing the histones through the major groove. It is illustrative to compare the magnitude of CpG methylation penalty with sequence dependent differences. Since there are roughly 1.5e88 possible 147 base pairs long sequence combinations (i.e., (4n+4(n/2))/2, n = 147), it is unfeasible to calculate all the possible sequence effects. However, using our elastic model we can provide a range of values based on a reasonably large number of samples. If we consider all possible nucleosomal sequences in the yeast genome (around 12 Mbp), the energy difference between the best and the worst sequence that could form a nucleosome is 0.7 kj/mol per base (a minimum of 1 kJ/mol and maximum of around 1.7 kJ/mol per base, the first best and the last worst sequences are displayed in Table S3 in Text S1). We repeated the same calculation for one million random sequences and we obtained equivalent results. Placing one CpG step every helical turn gives an average energetic difference between minor groove and major groove methylation of 15 kJ/ mol, which translates into ,0.5 kJ/mol per methyl group, 2 kJ/ mol per base for the largest effects. Considering that not all nucleosome base pair steps are likely to be CpG steps, we can conclude that the balance between the destabilization due to CpG methylation and sequence repositioning will depend on the PLOS Computational Biology | 4 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Figure 3. Methylated and non-methylated DNA elastic deformation energies. (A) Distribution of deformation energies for 147 bplong random DNA sequences with CpG steps positioned every 10 base steps (one helical turn) in minor (red and dark red) and major (light and dark blue) grooves respectively. The energy values were rescaled by the slope of a best-fit straight line of figure 2, which is 0.23, to source of circulating FGF-21. The lack of association between circulating and muscle-expressed FGF-21 also suggests that muscle FGF-21 primarily works in a local manner regulating glucose metabolism in the muscle and/or signals to the adipose tissue in close contact to the muscle. Our study has some limitations. The number of subjects is small and some correlations could have been significant with greater statistical power. Another aspect is that protein levels of FGF-21 were not determined in the muscles extracts, consequently we cannot be sure the increase in FGF-21 mRNA is followed by increased protein expression. In conclusion, we show that FGF-21 mRNA is increased in skeletal muscle in HIV patients and that FGF-21 mRNA in muscle correlates to whole-body (primarily reflecting muscle) insulin resistance. These findings add to the evidence that FGF-21 is a myokine and that muscle FGF-21 might primarily work in an autocrine manner. Acknowledgments We thank the subjects for their participation in this study. 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