Functions of Lr Genes beside Reduction of Puccinia triticina

Wheat leaf rust race specific resistance controlling genes (Lr) caused 0.01-0.03 decrease of SAGR = divided first two internodes with stem length. In addition, nitrogen transport in upper parts by those genes was 3-19% higher in comparison to near isogenic lines with nonspecific Lr 22a and Lr 22b genes. RAR (divided root nitrogen content with sum of up ground and root ones) values of Lr16, Lr29 and Lr37 between Lr1, Lr15 or Lr19 and nonspecific genes were explained by hydrolytic stability of endo-proteases responsible for cleaving of glutens disulphide bridges. According to RAR, Lr 34 seemed to be specific Lr gene but increased SAGR as was by nonspecific Lr genes focused SUTs as adequate for its conformity. Two different drought and heat stress respond systems were linked to Lr genes; one based on gluten degradation consequential through photosynthesis decrease and another on accelerated transfer of starch products and water in stem.


Introduction
Leaf rust causer Puccinia triticina is not the most destructive of wheat parasites.Stable appearance and large wheat growing areas have explained the responsibility for the major accumulated yield losses from single parasite in history (Chester 1946, Rijsdijk & Zadoks 1976).Considered by Khan et al. (1997), total yield could be reduced for about 1% for each 1% of infection, when higher temperatures begin earlier in the spring.In semiarid regions, grain yield was decreased by approximately 3.5% for each 10% of two last leaves average infection ( Jerkovic 2008).Until nowadays, 63 genes were proposed as Puccinia triticina reducing ones (Kolmer et al. 2010) and segregated as horizontal or vertical influential on parasites population reduction (Van der Plank 1963), to parasite races specific or nonspecific (Nelson 1978) and adult or seedling stage effective (Dyck 1991, Martinez et al. 2001, Saini et al. 2002).Highest reaction type (RT) was basis for partial resistance definition (Parlevliet & Van Ommeren 1975).Suspicion about huge number of different specific parasite host interactions predicted by gene-to-gene theory (Flour 1955(Flour , 1971) was articulated by Parlevliet (1988).Parlevliet (1986) recognized pleiotropic association between latency period (LP) and infection efficiency as well as LP was linked to specific resistance as its last character before overcome by changes in parasitic population ( Jerkovic et al. 1993, Jerkovic & Putnik-Delic 2004).Background influence on Lr genes expression was reduced by backcrossing with variety Thatcher (Lr NILs).Negative effects of genes for resistance on wheat genotype fitness (Van der Plank 1976) were described as lower yield of eight lines with genes introduced from relatives (The et al. 1988).Their activity was related to fungal elicitor (De Witt et al. 1993) while previously single glycoprotein in cytoplasm of susceptible variety, same as in the parasite cell wall as cause of lignifications was discovered (Koegel et al. 1988).The nature of nonspecific resistance characterized by decrease of infection efficiency was attempted to be explained across stomata number and function, leaf wax differences (Anker Ratar. Povrt. 50 :3 (2013) 6-12 & Nis 2000) or by analyses of early abortion (Niks 1981, Jacobs 1990, Niks & Dekens 1991, Jacobs & Kiriswa 1993).The inheritance of the trait was simple to polygenic (Parlevliet 1978, Lee & Shaner 1985, Dyck 1991, Das et al. 1992, Kolmer & Liu 2001).Across enhancements of the resistance, complementary Lr genes were recognized (Samborski & Dyck 1982, Jerkovic 1992, German & Kolmer 1992).The environmental influence on resistance genes expressions was involved in concept of interorganismal genetics (Loegering 1978) as well as of Lr ones summarised by Browder (1985).
The differences in growth were found to be related with leaf rust severity and regional grain yield potential and linked with nonspecific or adult stage resistance ( Jerkovic & Prijic 2009).From another side, it was discovered that Lr 16 and Lr 29, which were confirmed as race specific genes in interactions with parasite, had influence on carbohydrate fungicides accelerated degradation ( Jerkovic et al. 2012).It was known that the fungi cell wall was structured mostly from glycoproteins (Harder & Chong 1984) laminated by bridges same in one of basic elements (sulfur) within seed stored proteins (Shewry & Halford, 2002) or mentioned fungicides.Protein transport from seed began in units of molecular weight under 64 Kd continued in 24 Kd and finished below 10 Kd (Masson et al. 1986), so there was a room for their accelerated degradation.The presence of gluten in leaves was also noticed (Cumming et al. 2004), but their enzymatic degradation was not described.
According to previous states, the hypothesis was that specific Lr genes should cleave the disulphide bridges of seed stored proteins during germination visible through their distribution in organs of wheat seedlings, as well as that it will be of influence on growth.

Materials and Methods
Eighteen Lr near isogenic lines were sown in field nursery (susceptible border Novosadska rana 2) in semiarid region (around 600 mm 2 of the annual rainfall).Presented parasite severity on last two leaves was estimated in percents from the second ten-day period of May.Results from 2004 and 2011 were presented because of no or low antagonistic facultative parasites appearance.Two last internode and stem lengths estimated at the third last ten-day period of June 2010 and 2011 were divided and represented as stem growth ratio (SAGR).The second leaf length of seedlings was compared with the first one (LGR) ten days after emerging of 60 seeds sown in 2 dl pots with compost and grown in greenhouse on constant air temperature of 20 o C and 11 hours of day light regime, daily watered.Genotypes with near equal lengths of leaves were pronounced medium (M), those with higher second one for more than 1 cm were H 2 as opposite were L. Puccinia triticina severity on the last two leaves was presented in percents of the affected area.Lines for the tests divergent by nonspecific and specific resistance control were chosen across the specific resistance character, prolonged latency period in field and low reaction type (RT) according to scale by Stakman et al. (1962) to the races 2 and 77 ( Johnston & Browder, 1966) at seedling stage.Seed of Lr lines from same locality in 2010 were sown in 2 dl pots or 3 l cylinder filled by compost or pure sand and grown at the same regime as for LGR analysis.Two or three week old seedlings after emerging were cleared in water, completely dried and fine milled.Nitrogen content in root and up ground part was estimated in two replications by method previous used for seed protein evaluation (ICC method 1994).Root (RC) or app ground (AC) values multiplied by 5.7 were adequate to protein based structures amount presented in percent (RPC or APC).RC divided by AC and RC sum was RAR.

Results and Discussion
Differences according to SAGR were in the interval from 0.53 of Lr 1 in 2010 to 0.71 of Lr 12 in 2011.Values of tested genotypes were increased for 0.022 in average when specific genes were present in comparison to adequate nonspecific Lr gene containing NIL.The SAGR of Lr 12 line was higher for 0.01 than of Lr 22b, which could be explained by complementary effect with genes as were Lr 2c, Lr34, Lr 13 etc.SAGR of basic variety Thatcher was closest to Lr 2c line indicating different Lr genes accumulations in NILs.The average severity of Puccinia triticina was higher for 16% unexpected in more continuously dry year.Decreased severity of obligate parasite was related more to antagonism with Pyrenophora tritici repentis than to Septoria tritici ( Jerkovic 2008).The 5 Lr lines were of the same maximal rust severities 50 or 70 (SAGR 0.53-0.60)as another 5 were such but with values 40 or 50 (SAGR 0.67-0.71),also indicating the different nonspecific resistance genes effects in near isogenic lines beside race specific ones.Less maximally rusted than expected by SAGR where those with prolonged LP, visible through lower parasite development on last two leafs in period from the end of May (Table 1) and pronounced as race specific resistance genes carried like Lr 1, Lr 2a, Lr 16, Lr 19,Lr 21, Lr 29 and Lr 37. Decreasing of maximal severity of the parasitic attack was strong negative correlated to SAGR (r= -0.75 to -0.80).Without specific resistant genotypes recognized by lower initial infection severities, r-values were -0.81 to -0.86.Maximal severities of parasite attacks in two years was absolute correlated (r=0.98).Lower maximal intensity than on Thatcher was achieved by genes different positioned on chromosomes (McIntosh et al. 1995;Purnhauser et al. 2000).For confirmation of the above, recent dividing of Lr genes in two groups, the Lr 1 (5D) and Lr 2a (2D) genes were regional express able through lower RT at seedling stage to the particular races of pathogen (Boskovic & Browder 1976, Prijic & Jerkovic 2010), while generally most effective by RT decrease were Lr 19 (7D) and Lr 24 (3D) ( Jerkovic 1992); even chromosomes from D genome were described as suppressor genes containing (Dyck 1987, Nelson et al. 1997).Lr 1 and Lr 19 were recently proved as such according to lower RT (Tables 2 and 3).The nature of Lr 15 gene was not possible to discover according to analyses of regional host parasite interactions.Adequate race 162(A) proving its specific character was isolated in India (Gupta et al. 2008).
Genes like Lr 2, Lr 2b, Lr 3 or Lr 3b were SAGR increase able in comparison to basic Thatcher and much larger distanced from Lr 22b by the character than was achieved by specific resistance genes simultaneous presence with at last mentioned gene.Such, they were defined as adult plant or nonspecific resistance controlled genes with lower effect as were Lr 13 or Lr 48 and Lr 49 (Saini et al. 2002).Presence of such genes was followed by leaf tip necrosis (Schnurbrush et al. 2004, Mishra et al. 2005).However, near isogenic lines were multiple based when 2D chromosome was focused, where discovered genes were responsible for day light insensitivity (Worland & Law 1986) likely to be responsible for SAGR increase.For the difference Lr 34 was defined as non-hypersensitive (Rubiales & Niks 1995)  as adequate NIL was with higher SAGR, than was of Thatcher.Carbon flux discovered by Bolton et al. (2008) and lower RAR value in comparison to nonspecific resistance controlling genes were supplement to define unusual gene type.According to recent results Lr 34 was responsible for the realest of the seed content described by Aoki et al. (2006) and most likely to be sucrose transporter (SUT).Sugar, arabinose, xylose and mannose were adequate for haustorium mother cell formation in vitro, whereas only one hour of water lack during the initial infection was enough for its rejecting (Heat 1990).Enzymes for starch or soluble carbohydrates cleaving were weighted 45-59 Kd (Carneiro et al. 2004) and had to be transported last during heat and drought stress explaining different colours of leaf tips (K and Ca).Their optimum at higher temperatures in acid environment (Halverson & Barry 2003) was adequate to the state.RAR values of Lr 1, Lr 15, Lr 19 were equal and distanced from Lr 22b line more than Lr 16, Lr 37 and Lr 29.The active sites of adequate enzymes could not be the same also confirmed by different RTs when mono pustule isolate of parasite was applied (Boskovic & Browder 1976, Jerkovic 1992, Mesterhazy et al. 2000, Bulos et al. 2006, Winzeler et al. 2000, Prijic & Jerkovic 2010).Simulated results when RAR values sum was equalized with seven defined those genes as responsible for 3 to 19% of root proteins lack.Results confirmed enough energetic level of all tested enzymes to cleave disulphide linkage of gluten (Tables 2 and  3).Prolamins were of remarkable diversity in size, gliadin, glutenin ratio, frame shift mutations and sulfur content (Nagy et al. 2005).Total of 35-40% of wheat gluten was gliadin characterized by intra chains cross links so called bounds, branches or bridges, as well as such amounted were intra and extra bounded glutenin (Wall 1971).Gliadin first reached their proportion in seed (Wrigley et al. 1980) and indicated the external bounds as the primary target of hydrolysis or enzymes.The protein disulfide isomerases (PDI) were responsible for cysteine join in covalently different polypeptides or stabilization of protein via bounds (Ciaffi et al. 1999).Primarily the gliadin monomers were transported to vesicles via Golgi apparatus (GA) according to Rubin et al. (1992).Bound producing effect of PDI enzymes was linked to maturating conditions characterized by higher temperatures and drought.Fructose-1, 6-diphosphatase from spinach leaf chloroplasts weighted 92-115 Kd, while Nitrogen and other nutritives from compost were not absorbed in above described growth phases according to Vuurde & Tonneyck (1978).Identified genes in hard red spring wheat (Oelke & Kolmer 2004, 2005, Martinez et al. 2007) as in soft spring ones (Kolmer 2003, Wanishe & Millus 2004) were in agreement with achieved protein contents in seed, as well as state that genes for gluten synthesis were not linked with Lr 19 (Slikova et al. 2003).In addition, there was no relation between protein content and RAR values according to its same result as was of Lr 1 line.
For the digression, hypersensitive reaction was related to same mechanism with different source of sulphur from fungi cell wall or membrane instead of gluten.Sulphur dioxide, which was expected to be consequence of enzymes produced by specific Lr genes, was transferred in acid forms when reached chloroplast while fall of PH below 6.5 caused chlorophyll degradation ( Jolivet et al. 1992).

Conclusions
According to recently presented results, genes controlling race specific resistance to Puccinia triticina were particular endoprotease also responsible for gluten disulphide bridges degradation consequential through increased transfer of proteins in plant up ground parts from seed, decreased SAGR and increased LGR in comparison to Lr 22b line.Enzymes were with different hydrolytic stability according to different effects to the gluten.Other type of Lr genes cause of increased SAGR were most likely related to starch and its products degrading and had no influence on RAR.For the difference of both above-mentioned gene groups, according to slightly decreased RAR and SAGR, increase of Lr 34 was adequate to transporters enzymatic family.However, two different drought and heat stress respond mechanisms were discovered.When race nonspecific Lr genes were present, plant growth was because of below parts followed by their rejection.Vice versed, specific Lr genes reduced photosynthesis and across locally more viable water prevented starch and gluten formation allowing further growth of recently present organs consequential through prolonged total leaf area duration and equilibrated growth.
Recently proposed method could be used for discovering of Lr genes without interactions with parasite, but did not signal the opportunity for their precise identification.

Table 2 .
Reaction to Puccinia triticina races, LGR and nitrogen distribution of three-week-old seedlings

Table 3 .
Reaction to Puccinia triticina races, LGR and nitrogen distribution of two-week-old seedlings